PNPLA3 as a driver of steatotic liver disease: navigating from pathobiology to the clinics via epidemiology
0 
, Abstract
Steatotic liver disease (SLD), particularly metabolic dysfunction-associated SLD, represents a significant public health concern worldwide. Among the various factors implicated in the development and progression of this condition, the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene has emerged as a critical player. Variants of PNPLA3 are associated with altered lipid metabolism, leading to increased hepatic fat accumulation and subsequent inflammation and fibrosis. Understanding the role of PNPLA3 not only enhances our comprehension of the pathomechanisms driving SLD but also informs potential therapeutic strategies. The molecular mechanisms through which PNPLA3 variants contribute to lipid dysregulation and hepatocyte injury in SLD are critically discussed in the present review article. We extensively analyze clinical cohorts and population-based studies underpinning the association between PNPLA3 polymorphisms and the risk of developing SLD, and its liver-related and protean extrahepatic outcomes, in concert with other risk modifiers, notably including age, sex, and ethnicity in adults and children. We also discuss the increasingly recognized role played by the PNPLA3 gene in liver transplantation, autoimmune hepatitis, and acquired immunodeficiency syndrome. Finally, we examine the clinical implications of PNPLA3 diagnostics regarding risk stratification and targeted therapies for patients affected by SLD in the context of precision medicine approaches.
Keywords
INTRODUCTION
The global increase in the prevalence of liver diseases, particularly Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), has become a significant public health issue. MASLD is characterized by an excessive accumulation of fat in the liver. Globally, the adult population has witnessed an increased prevalence of MASLD from 25.3% in 1990-2006 to 38.2% in the years 2016-2019[1]. MASLD identifies steatosis associated with ≥ 1 factor of cardiometabolic risk[2]. Additionally, MASLD is closely associated with either the full metabolic syndrome or its components (e.g., obesity, type 2 diabetes) and often with the complications of the metabolic syndrome (e.g., cardio-nephro-vascular disease)[3]. Given its rising prevalence and potential to progress to more severe forms such as Metabolic Dysfunction-Associated Steatohepatitis (MASH), cirrhosis, and Hepatocellular Carcinoma (HCC), understanding the underlying mechanisms driving MASLD is crucial for the prognosis of patients with MASLD and for developing effective prevention and treatment strategies[4]. Particularly, the Patatin-like phospholipase domain-containing protein 3 (PNPLA3) has garnered significant attention in the pathogenesis of steatotic liver disease due to its strong association with hepatic fat accumulation and progression to MASLD[5]. Genetic variants of PNPLA3, particularly the I148M polymorphism, have been associated with increased susceptibility to liver damage and fibrosis[6,7]. This makes PNPLA3 a key focus for understanding the molecular mechanisms underlying steatosis and potential therapeutic targets for managing liver diseases. Additionally, its role in lipid metabolism further emphasizes its importance in liver health and disease.
Here we try to offer an in-depth overview of PNPLA3 as a driver of Steatotic Liver Disease (SLD) by exploring both pathobiological mechanisms and epidemiological evidence. By synthesizing current knowledge on how this gene contributes to hepatic fat accumulation and its implications for public health policies aimed at managing rising rates of MASLD globally, we aim to shed light on future research directions that could pave the way for innovative treatments in the setting of precision medicine approaches.
ROLE OF THE PNPLA3 GENE IN HEALTH AND STEATOTIC LIVER DISEASE
Among the various genetic factors influencing MASLD, the patatin-like phospholipase domain-containing protein 3 (PNPLA3, OMIM: 609567) gene has garnered significant attention. The gene, also known as adiponutrin, calcium-independent phospholipase A2ε (iPLA2ε), acylglycerol transacylase, or 1-acylglycerol-3-phosphate O-acyltransferase, was first identified in mouse 3T3 pre-adipocyte cell lines and located on human chromosome 22q13.1 by sequence similarity search[8]. It belongs to the family of patatin-like phospholipase domain-containing proteins (PNPLAs) that in humans consists of nine members with crucial roles in preserving the structure and function of organelle membranes, cell growth, signaling, cell death control, and the general metabolism of lipids, including triacylglycerol, phospholipids, ceramides, and retinyl esters [Table 1][9].
Characteristics of the human patatin-like phospholipase domain-containing proteins
| Member | OMIM | Alias | Chromosomal localization* | Exons | Transcript length (nt)/acc. no** | Protein size (aa)/acc. no.** | Mw (kDa)*** | Selected function |
| PNPLA1 | 612121 | Omega-hydroxyceramide transacylase; FLJ38755;ARCI10; dJ50J22.1; EC 2.3.1.296 | 6p21.31 | 11 | 2367 NM_173676.2 | 437 AAI03906.1 | 48.96 | Transfers fatty acyl groups from triacylglycerol to omega-hydroxy ceramides to form acylceramides |
| PNPLA2 | 609059 | Adipose triglyceride lipase (ATGL); Desnutrin; Transport-secretion protein 2 (TT2); FP17548; Pigment epithelium-derived factor receptor; Phospholipase A2, calcium-independent | 11p15.5 | 10 | 2416 NM_020376.4 | 504 NP_065109.1 | 55.32 | Triglyceride lipase; catalyzes the first step in the hydrolysis of triglycerides in adipose tissue |
| PNPLA3 | 609567 | Adiponutrin (ADPN); Phospholipase A2, calcium-independent ε; C22orf20; 1-Acylglycerol-3-Phosphate O-Acyltransferase; Lysophosphatidic acid acyltransferase; Acylglycerol transacylase | 22q13.31 | 9 | 2753 NM_025225.3 | 481 AAH65195.1 | 52.84 | Multifunctional enzyme with both triacylglycerol lipase and acylglycerol O-acyltransferase activity |
| PNPLA4 | 300102 | GS2 Gene (GS2); Phospholipase A2, calcium-independent η (IPLA2-eta); DXS1283E | Xp22.31 | 8 | 3342 NM_004650.3 | 253 AAH20746.1 | 27.88 | Enzyme that has both triacylglycerol lipase and transacylase activities |
| PNPLA5 | 611589 | GS2-like protein (GS2L); DJ388M5.4; DJ388M5 | 22q13.31 | 9 | 2540 NM_138814.4 | 429 AAH31820.1 | 47.91 | Abundant triacylglycerol hydrolase activity |
| PNPLA6 | 603197 | Neuropathy target esterase (NTE); Neurotoxic esterase; SPG39; Sws; NTEMND; BNHS, LNMS, OMCS | 19p13.2 | 37 | 4536 NM_001166111.2 | 1375 NP_001159583.1 | 146,215.65 | Deacetylates intracellular phosphatidylcholine to produce glycerophosphocholine |
| PNPLA7 | 612122 | NTE-Like1 (NTEL1); Chromosome 9 open reading frame 111 (C9org111); NTE-R1; RP11-48C7.2; FLJ43070; FLJ31318; FLJ44279 | 9q34.3 | 37 | 4675 NM_001098537.3 | 1342 NP_001092007.2 | 148,431.39 | Endoplasmic reticulum transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine |
| PNPLA8 | 612123 | PNPLA-γ, Phospholipase A2, calcium-independent, intracellular membrane-associated γ (IPLA2-gamma); IPLA2-2; MMLA | 7q31.1 | 15 | 4681 NM_015723.5 | 782 AAH32999.1 | 88,476.86 | Cleavage of fatty acids from phospholipids, thereby regulating membrane physical properties and the release of lipid second messengers and growth factors |
| PNPLA9/PLA2G6 | 603604 | Phospholipase A2, group VI (PLA2G6), Phospholipase A2, calcium-independent (IPLA2); Phospholipase A2, calcium-independent, group VI, A (IPLA2-VIA); PARK14, Neurodegeneration with brain iron accumulation 2 (NBIA2); 85/88 kDa calcium-independent phospholipase A2; 2-Lysophosphatidylcholine acylhydrolase; Palmitoyl-CoA hydrolase; CaI-PLA2; GVI PLA2; INAD1; GVI | 22q13.1 | 18 | 3299 NM_003560.4 | 806 AAH36742.2 | 89,903.01 | Hydrolyses membrane phospholipids to produce potent lipid second messengers |
The individual members of this protein family vary in size; the smallest member, PNPLA4, consists of 253 amino acids, while the largest member, PNPLA6, contains 1,327 amino acids [Figure 1][9]. These members are further classified into the adiponutrin group, which includes PNPLA1-5, the neuropathy target esterase group, including PNPLA6 and PNPLA7, and PNPLA8 and PNPLA9 which have specific characteristics and do not belong to either of these two groups[9].
Figure 1. Structure of human patatin-like phospholipases. The family of PNPLAs consists of 9 members (PNPLA1-PNPLA9) characterized by a patatin-like phospholipase domain. The first five members are subclassified into the adiponutrin (ADPN) group, while members PNPLA6-PNPLA7 belong to the neuropathy target esterase (NTE) group, and the last two members do not belong to either group. The PNPLA motif of PNPLA2-PNPLA9 typically contains a GXGXXG motif, a GXSXG motif, and a DGA/G motif. The active nucleophile site in the GXSXG motif and the proton acceptor site in the DGA/G motif are marked in red letters. Members of the NTE groups also have several cAMP/cGMP binding site motifs (CNMP-Bind), while PNPLA9 contains four ankyrin repeats. The numbers correspond to amino acid positions in human proteins. Motif search was done using the Expasy ScanProsite tool (https://prosite.expasy.org/scanprosite/) and protein sequences of individual PNPLA proteins were taken from GenBank sequence (https://www.ncbi.nlm.nih.gov/protein/) entries listed in Table 1.
PNPLA3, located on the long arm of chromosome 22, encodes a transmembrane protein with triglyceride hydrolase activity that plays a pivotal role in lipid metabolism within hepatocytes[10]. Variants of this gene, particularly the I148M polymorphism (rs738409), have been linked to increased hepatic steatosis and higher odds of hepatic injury [Figure 2][11]. This single nucleotide polymorphism (SNP) leads to an amino acid substitution that alters the enzymatic function of PNPLA3, impacting triglyceride hydrolysis, lipid droplet-Golgi dynamics, mitochondrial dysfunction, retinol metabolism, antioxidant responses, and increased TGF-β1 signaling, thereby contributing to lipid accumulation in the liver[11-13]. The reasons for these changes that occur in the absence of functional PNPLA3 will be discussed later (see Section "PNPLA3-I148M gene variant: implications for triglyceride hydrolysis"-"PNPLA3-I148M and retinol metabolism"), offering a comprehensive understanding of the underlying mechanisms involved.
Figure 2. The PNPLA3 gene. The PNPLA3 gene is located on the long arm (q-arm) of the human chromosome in region 22q13.31. It encodes a protein of 481 amino acids in size that has a PNPLA motif (underlined), which contains the characteristic GXGXXG, GXSXG, and DGA/G motifs (all in blue). The non-synonymous substitution (rs738409) of isoleucine to methionine at position 148 results in a protein with reduced enzymatic activity. The image of the ideogram was taken from the Genome Data Viewer of the National Library of Medicine (https://www.ncbi.nlm.nih.gov/gdv/).
Research over the past decade has elucidated several mechanisms through which PNPLA3 influences hepatic steatosis. The I148M variant appears to impair lipolysis and promote lipid droplet formation within hepatocytes. This dysfunction not only results in increased triglyceride storage but also triggers inflammatory pathways that can lead to cellular injury and fibrosis over time[12]. Understanding these pathobiological processes is essential for identifying potential therapeutic targets. Studies on human hepatocytes with PNPLA3-148I and -148M variants implanted in the livers of immunodeficient chimeric mice have shown that hepatocytes carrying the PNPLA3-148M variant, whether from homozygous donors or overexpressed in a heterozygous background, showed more severe microvesicular steatosis and ballooning degeneration compared to those with the 148I variant. This indicates a heightened risk for steatohepatitis[14].
As extensively discussed in Section "ROLE OF THE PNPLA3 GENE IN HEALTH AND STEATOTIC LIVER DISEASE" of the present review, epidemiological studies have shown a strong link between PNPLA3 variants and susceptibility to MAFLD in various populations[15-18]. In obese individuals, the expression of PNPLA3 in the liver was higher in women than in men, correlating with estrogen levels. Estrogen receptor-α (ER-α) agonists increased PNPLA3 expression in human hepatocytes and liver organoids[18]. Researchers identified an ER-α-binding site within a PNPLA3 enhancer that drives the upregulation of the p.I148M variant through chromatin immunoprecipitation and luciferase assays, as well as CRISPR-Cas9 genome editing. This ultimately leads to steatogenesis and fibrogenesis in three-dimensional spheroids containing hepatic stellate cells (HSCs), indicating that the interaction between ER-α and the PNPLA3 p.I148M variant is a key player in the development of SLD in women[18].
Despite considerable advances in our understanding of PNPLA3's role in SLD, there are still several gaps in our knowledge base. For example, while much research has focused on its genetic implications, there is limited insight into how environmental factors, such as diet and lifestyle, interact with genetic predispositions to influence disease outcomes. Additionally, questions about how PNPLA3-related pathways can be targeted therapeutically remain largely unanswered. Some of these research questions will be addressed in the next sections of this review, specifically examining how an improved understanding of the role of PNPLA3 in the initiation and worsening of SLD due to various etiologies has opened avenues for targeted therapeutic interventions and lifestyle modifications aimed at mitigating liver damage.
PNPLA3-I148M gene variant: implications for triglyceride hydrolysis
PNPLA3 is involved in lipid metabolism, facilitating the hydrolysis of triglycerides into free fatty acids and glycerol. The missense mutation at amino acid position 148, which changes isoleucine to methionine in PNPLA3, is located next to the Ser47-Asp166 catalytic dyad and is part of a hydrophobic substrate-binding groove in the active site[19]. When overexpressed in the liver of mice, PNPLA3-I148M caused a significant increase in the number and size of lipid droplets, as well as in the levels of triglycerides and cholesterol esters in the tissue[19]. The authors of the study also showed that the rise in triglycerides is due to a decrease in hydrolysis rather than an increase in fatty acid esterification[19].
Recent findings suggest that PNPLA3 preferentially hydrolyzes polyunsaturated triglycerides in an adipose triglyceride lipase (ATGL)-independent manner, mobilizing polyunsaturated fatty acids for phospholipid desaturation and increasing hepatic secretion of large-sized very low density lipoproteins [Figure 3][20]. However, when mutated, this enzymatic function is compromised, leading to the accumulation of triglycerides within hepatocytes, causing SLD.
Figure 3. Enzymatic activity of PNPLA3. PNPLA3 is an important protein that catalyzes crucial reactions in lipid metabolism. It primarily catalyzes three reactions: 
acting as a triacylglycerol lipase, 
functioning as a phospholipase A2 by removing the fatty acid attached to the 2-position of phosphatidylethanolamine, choline plasmalogen, and phosphatides, and 
specifically catalyzing coenzyme A (CoA)-dependent acylation of 1-acyl-sn-glycerol 3-phosphate (2-lysophosphatidic acid) to generate phosphatidic acid.
In summary, the PNPLA3-I148M variant exhibits decreased enzymatic activity in metabolizing triglycerides and is also less active in secreting hepatic triglycerides. Consequently, this variant negatively impacts the balance between hepatic triglyceride metabolism, storage, and secretion.
Effects of PNPLA3-I148M on lipid droplet-Golgi dynamics
The Golgi apparatus plays a significant role in processing lipids for secretion or incorporation into membranes[21]. Under normal circumstances, PNPLA3 interacts dynamically with lipid droplets during triglyceride hydrolysis, allowing for efficient transfer of lipids between droplets and other cellular compartments, including the Golgi apparatus. The accumulation of lipids disrupts normal lipid size and composition, affecting their ability to interact efficiently with Golgi membranes. This disruption may also provoke dyslipidemia and trigger stress responses. Recent work demonstrated that the mutated PNPLA3-I148M variant induces direct structural alterations in the Golgi apparatus, including increased lipid droplet-Golgi contact sites and enlarged Golgi cisternae[10]. Importantly, these changes were associated with morphological, proteomic, and transcriptional changes within the cell that are compatible with those found in the MASH spectrum. This supports the notion that the I148M mutation alone is capable of driving all stages of MASLD[22]. Another report demonstrated that PNPLA3 can physically interact with the lipid-droplet-associated protein Perilipin 5 (PLIN5) and the adipose triglyceride lipase (ATGL), the rate-limiting enzyme in lipolysis[22]. The binding of either PNPLA3 or PLIN5 to ATGL reduces its lipogenic activity. Interestingly, compared to PNPLA3, the binding of PNPLA3-I148M to ATGL exhibited a stronger inhibitory effect[22]. Since once activated, ATGL facilitates the transfer of lipids to the Golgi apparatus for further processing, reduced activation of ATGL will lead to impaired Golgi dynamics, affecting overall lipid homeostasis[22].
Role of PNPLA3-I148M in mitochondrial dysfunction
In a recent study by Gou et al., it was demonstrated that the non-synonymous PNPLA3-I148M substitution provokes free cholesterol accumulation in human hepatic stellate cell (HSC) line LX-2 by reducing the expression of the ATP-binding cassette sub-family G member 1 (ABCG1) and inhibiting cholesterol efflux[12]. The accumulation of cholesterol within cells further impairs mitochondrial structure and functionality, resulting in a reduced expression of proteins associated with mitochondria, including superoxide dismutase (SOD-1) and the mitochondrial protein Mitofusin (MFN2), which plays a central role in regulating mitochondrial fusion and cell metabolism. This process ultimately stimulates the activation of LX-2 cells and contributes to the emergence of a fibrotic phenotype[12]. These results offer fresh insights into how PNPLA3-I148M influences lipid metabolism, mitochondrial impairment, and liver fibrosis. In the same line, PNPLA3-I148M was linked to respiratory chain complex IV insufficiency, elevated secretion of reactive oxygen species (ROS), and reduced expression of the orphan nuclear receptor NR4A1, which regulates the ROS/endoplasmic reticulum stress pathways[13,23].
Consequently, individuals with homozygous PNPLA3-I148M mutations exhibit changes in intrahepatic anabolic and catabolic processes, as well as mitochondrial function. These alterations include a reduction in de novo lipogenesis whereas intrahepatic mitochondrial beta-oxidation and ketogenesis are increased[24]. Such changes are associated with an elevated mitochondrial redox state and a decreased flux of hepatic mitochondrial citrate synthase. These findings confirm that SLD caused by the PNPLA3-I148M variant results in dysfunctional liver mitochondria[24].
Modulation of TGF-β1 signaling by PNPLA3-I148M
PNPLA3 not only impacts aspects of fat metabolism in the liver but also has a direct profibrogenic activity[25]. The expression of the PNPLA3 gene and protein rises during the initial stages of activation and remains elevated in fully activated HSCs, while silencing PNPLA3 notably reduces the levels of the profibrogenic protein α-smooth muscle actin[25]. Primary human HSCs with the I148M variant exhibit significantly increased expression and release of proinflammatory cytokines and show lower retinol levels but a higher accumulation of lipid droplets. Similarly, LX-2 cells that stably overexpress I148M demonstrate enhanced proliferation and migration, reduced retinol levels, diminished transcriptional activities of retinoid X receptor/retinoid A receptor, and increased lipid droplet formation. Silencing PNPLA3-I148M leads to decreased expression of collagen1α1, suggesting that PNPLA3 is essential for HSC activation and that its genetic variant I148M enhances the profibrogenic characteristics of HSCs, elucidating a molecular mechanism underlying the increased risk for progression and severity of liver diseases in patients carrying the I148M variant[25]. In this context, it is worth noting that in primary human HSC, PNPLA3 is upregulated by TGF-β1 and that the mutant PNPLA3, but not the wild type PNPLA3 protein, reduces the secretion of matrix metalloprotease (MMP) 2 (MMP2) that suppresses collagen type I expression and tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2) that promote fibrosis in the injured liver by inhibiting MMPs[26-28]. Contrastingly, another study has shown that the downregulation of PNPLA3 resulted in increased expression of profibrogenic factors and exacerbated fibrotic responses in human HSCs in the presence of TGF-β1, regardless of the PNPLA3 genotype[29].
PNPLA3-I148M and retinol metabolism
Liver extracts from individuals homozygous for the PNPLA3-I148M minor allele exhibit increased concentrations of retinyl palmitate but a decreased retinol-to-retinyl-palmitate ratio[30]. These variations were still significant in a multivariate analysis that accounted for the severity of steatosis. Additionally, the levels of minor retinyl-fatty acid esters were also found to be elevated in those carrying two copies of the PNPLA3-I148M variant[30]. Excess of retinoids is linked to hepatic fibrosis, and the loss of intracellular retinoid stores is a hallmark of HSC activation[31,32]. This supports a possible association between the PNPLA3 variant, hepatic retinoid metabolism, SLD, and other types of chronic liver disease in humans. Another report found that the PNPLA3-I148M genotype is a strong predictor of circulating retinol-binding protein 4, a reliable surrogate of retinol concentrations in humans[33], underpinning the important role of PNPLA3-I148M as a crucial lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans, a crucial factor associated with the initiation and progression of liver disease. Another study reported that carriers of the mutant allele with SLD and obesity or obesity alone had reduced concentrations of fasting retinol[34], again linking PNPLA3-I148M to a feature associated with hepatic steatosis.
CLINICAL EPIDEMIOLOGY
PNPLA3 variants have a significant impact on the development, progression, and complications of MASLD, affecting both liver-related and extrahepatic manifestations in adults and children. To support this claim, we will explore the various ways in which the PNPLA3 gene interacts with ethnicity, population-based epidemiology, and clinical epidemiology. Furthermore, we will discuss how the PNPLA3 gene interacts with other gene variants, sex, and liver disease among those living with human immunodeficiency virus (HIV) infection, or in the context of liver transplantation, or in autoimmune hepatitis (AIH).
PNPLA3: race/ethnicity, and spectrum of liver disease
Although the concepts of “race” and “ethnicity” are subjective constructs that lack a universally accepted definition, several studies have identified racial/ethnic disparities in MASLD[35]. These differences among ethnic groups can provide insight into various aspects of SLD, such as access to care and pathogenic determinants. Caldwell was one of the first investigators to note that MASH and cryptogenic cirrhosis (referred to as “burnt-out MASH”) were underreported among African Americans. He pointed out that this discovery contradicted the overrepresentation of major risk factors for MASH among this population. Caldwell suggested that this discrepancy could be due to under-recognition, under-referral, or a lower prevalence of these disorders among African Americans[36]. We now understand that, in addition to lifestyle habits and varying access to care, the risk of MASLD in each population tends to align with the frequency of the G allele of the PNPLA3 gene in that population[35,37]. Supporting this idea, a recent global assessment of disability-adjusted life years and deaths in 2021 identified Mexico as the country with the highest relative MASLD burden worldwide[38]. This finding is consistent with another study showing that Hispanics with Mexican, Central, and South American heritage have a higher prevalence of the PNPLA3-G risk allele compared to Hispanics of European or Afro-Caribbean descent[39]. Table 2 summarizes the PNPLA3-G risk allele frequency in different countries and geographical areas[6].
PNPLA3-G risk allele frequency in different countries and geographical areas1
| Country/geographical areas | G allele frequency (%) |
| Sub-Saharan Africa | 12 |
| Europe | 23 |
| Dominican Republic | 25 |
| South Asia | 24-30 |
| East Asia | 35-45 |
| Central and South America | ~50 |
| Mexico | 52 |
| Guatemala | 69 |
| Peru | 72 |
The differences in the frequency of the PNPLA3-148 M allele [Table 2] likely contribute - along with variations in obesity prevalence, urbanization levels, diet, and other lifestyle habits - to the observed differences in MASLD prevalence in the same countries and geographical areas[6].
Concerningly, the PNPLA3-G allele is linked to markers of liver fibrosis[39], cirrhosis[40], liver-related events, and mortality[41], as well as HCC in Caucasians[42]. It is crucial to note that the development of MASLD is complex, and other risk modifiers, including body mass index (BMI), gut microbiota, and mitochondrial genetics, interact with PNPLA3 gene variants[43,44].
Finally, it should be emphasized that PNPLA3 risk alleles are strong predictors of disease worsening in individuals with MASLD, as well as in those with alcohol-associated liver disease and HCV[45]. Conversely, PNPLA3 does not seem to contribute to the risk of HCC in individuals with HBV infection[46], probably because steatosis may hamper HBV replication cycle[47].
PNPLA3 gene variants affect liver-related outcomes in MASLD
A consistent line of research [Table 3][4,18,41,48-65] strongly supports the notion that risk variants of the PNPLA3 gene significantly contribute to increasing the odds of MASLD development[56] and interact with the host’s features (e.g. age, sex, reproductive status, and visceral adipose tissue) to increase the odds of fibrotic progression of MASLD to cirrhosis and its complications[18,52,57,59] accounting for the increased risk of liver-related events[58,59]. Additionally, obesity and excessive alcohol drinking strongly potentiate the risks of liver cirrhosis, hepatocellular carcinoma, and mortality due to hepatic disorders[53].
Role of PNPLA3 gene variants in liver-related outcomes
| Author, year [Ref.] | Method | Findings | Conclusions |
| Lisboa et al., 2020[48] | This study recruited 148 subjects with MASLD, of whom 54 had biopsy-proven MASH and 94 had simple steatosis, as well as 137 HCs | In a fully adjusted multivariable model, the G allele was associated with higher risks of MASLD (OR = 1.69, 95%CI: 1.21-2.36, P = 0.002) and MASH (OR = 3.50, 95%CI: | The G allele is associated with more severe liver histology |
| Idilman et al., 2020[49] | 174 patients with biopsy-proven MASLD and 151 HCs | After adjustment for confounding variables, the GG genotype was strongly associated with significant hepatic fibrosis (aOR = 3.031, P = 0.012) | The PNPLA3 GG genotype is a risk for more severe MASLD |
| Grimaudo et al., 2020[41] | 471 MASLD subjects were followed for a median time of 64.6 months | After adjusting for confounding factors, PNPLA3 C>G variant was linked to higher odds of hepatic decompensation, HCC, and liver-related death at multivariate Cox regression analysis [HR], 2.10, 95%CI: 1.03-4.29; P = 0.04; HR, 2.68, 95%CI: 1.01-7.26; P = 0.04; HR, 3.64, 95%CI: 1.18-11.2; P = 0.02, respectively. These results were confirmed in the subset of 162 individuals with AF/cirrhosis | MASLD subjects carrying the PNPLA3 rs738409 G>C variant are exposed to the risks of liver-related events and death |
| Pennisi et al., 2020[50] | 430 and 342 patients in whom FIB-4 and LSM were available at the baseline and at the last follow-up visit, respectively, were enrolled | Fibrosis progression was observed in 8.1%, 13.2%, and 23.2% of patients with PNPLA3 CC, CG, and GG genotypes, respectively (P = 0.03), regardless of confounding factors (OR, 1.90, 95%CI: 1.05-3.42; P = 0.03) Sensitivity analyses confirmed the PNPLA3 variant as a strong predictor of fibrosis worsening by both FIB-4 (OR, 2.28, 95%CI: 1.22-4.24; P = 0.009) and LSM (OR, 2.11, 95%CI: 1.13-4.42; P = 0.01) in patients followed for up to 90 months | PNPLA3 rs738409 C>G variant independently predicts the worsening of |
| Salari et al., 2021[51] | Meta-analytic review was conducted on 31 published studies, totaling 9,973 cases and 13,048 controls | The CC genotype has a reduced risk of MASLD (OR = 0.48, 95%CI: 0.40-056), while CG (OR 1.19, 95%CI: 1-1.33) and GG genotypes (OR 2.05, 95%CI: 1.64-2.56) were at increased MASLD risk | The CC genotype is 52% less prone to the risk of developing MASLD, while the CG genotype is 19% more exposed to the risk of developing MASLD. Finally, the GG genotype carries a 105% higher odds of MASLD |
| Li et al., 2022[52] | 523 Chinese with biopsy-proven MASLD. VFA was assessed with BEI | For any given level of VFA, the risk of SF was greater among those with the rs738409 G genotype and VFA remained significantly associated with SF only among those with the rs738409 G-allele | PNPLA3 rs738409 G and VFA interact to increase the risk of SF |
| Kim et al., 2022[53] | Prospective study of 414, 209 participants who had no previous diagnosis of cirrhosis and HCC at the baseline and were followed for up to 5 years | Compared to non-obese non-excessive drinkers and noncarriers of the PNPLA3-I148M variant, individuals with obesity, excessive drinking, and homozygous carriers of the PNPLA3-I148M variant exhibited highly increased odds of cirrhosis (aHR 17.52, 95%CI: 12.84-23.90), HCC (aHR, 30.13, 95%CI: 16.51-54.98) and mortality owing to hepatic causes (aHR, 21.82, 95%CI: 13.78-34.56) | The PNPLA3-I148M variant status enhances the odds of cirrhosis, HCC, and liver-related mortality among subjects with obesity and high alcohol consumption |
| Chen et al., 2023[54] | Participants from two unrelated cohorts, MGI (n = 7,893) and UK Biobank (n = 46,880), were enrolled. In these cohorts, MASLD was defined as raised ALT values after excluding competing etiologies of CLD. Values of 1.3-2.67 defined an “indetermined FIB-4 score”, while high-risk FIB4 scores were defined by values > 2.67 | Among those who had indeterminate FIB4 scores, individuals with T2D and the PNPLA3 rs738409-GG genotype had a cirrhosis incidence rate in the same order of magnitude as those with high-risk FIB4 scores and 2.9-4.8 times higher than patients with T2D but CC/CG genotypes Conversely, FIB4 < 1.3 was associated with a significantly lower risk of incident cirrhosis compared to that of those with high-risk FIB4 scores, irrespective of clinical risk factors and PNPLA3 risky genotype | PNPLA3 rs738409 and T2D can be used to identify MASLD subjects who, although currently considered at indeterminate risk, have a cirrhosis risk like those with FIB4 values considered high-risk |
| Koo et al., 2023[55] | 302 individuals with biopsy-proven MASLD were included in the study, with a median follow-up of 54 months. The primary outcome was a composite of LSM 9.6 kPa during the follow-up (for those subjects exhibiting F0-2 at the baseline), and ΔLSM ≥ 20% compared to baseline (for those individuals exhibiting F3-4 at the baseline) | The G allele in PNPLA3 rs738409, along with MASH at baseline, was associated with an increased risk of the primary outcome during follow-up regardless of confounding factors (HR per 1 risk allele, 2.08; 95%CI 1.45-2.99) | The G allele in PNPLA3 rs738409 increases the odds of fibrosis progression in MASLD |
| Zhao et al., 2023[56] | Meta-analytic review of 20 studies totaling 3,240 patients | A significant increase in association was found between rs738409 and MASLD across 5 different models | PNPLA3 rs738409 plays a major role in increasing the risk of MASLD |
| Rosso et al., 2023[57] | Retrospective analysis on 756 consecutive biopsy-proven European MASLD patients | After stratifying for age, sex, and BMI, a higher risk of LRE was observed in the subgroup of non-obese women over 50 years with the PNPLA3 GG risk genotype (log-rank test, | Compared to the wild-type allele (CC/CG), non-obese postmenopausal women with MASLD and the PNPLA3 GG risk genotype exhibit an increased risk of LRE |
| Seko et al., 2023[58] | 1,550 Japanese subjects with biopsy-proven MASLD were recruited and followed for a median of 7.1 years | In multivariate analysis, PNPLA3 CG/GG [HR] 16.04, P = 0.006) and FIB-4 index > 2.67 (HR 10.70, P < 0.01) independently predicted LRE | The PNPLA3-G allele carries an increased risk of LRE |
| Cherubini et al., 2023[18] | This study is based on three study cohorts: the Liver Biopsy Cohort (n = 1,861 European individuals submitted to liver biopsy for suspected MASH); an independent case-control cohort of severe MASLD (n = 4,374); and the population-based UK Biobank cohort (n = 347,127) | A specific multiplicative interaction occurring in women with PNPLA3-I148M determines SLD in at-risk individuals (steatosis and fibrosis, P < 10-10 advanced fibrosis/HCC, | A synergistic interaction between the female sex and the PNPLA3-I148M variant determines all stages of SLD, which is worse after the decline of estrogens |
| Chalasani et al., 2024[59] | 2,075 adults with biopsy-proven MASLD were followed for a mean time of 4.3 years | The independent predictors of MALO included PNPLA3-G, AF, age > 60 years, and T2D Among individuals with AF, those who have the G-allele had the highest cumulative incidence of MALO PNPLA3 and MALO were significantly more strongly associated among those > 60 years, women, and those with AF or T2D (sHR: 2.1, 95%CI: 1.5-2.8; sHR: 1.4, 95%CI: 1.1-1.9; sHR: 1.9, 95%CI: 1.5-2.4; sHR: 2.1, 95%CI: 1.5-2.8) | AF, age, T2D, and sex interact with PNPLA3 rs738409, contributing to worsening the risk of MALOs |
| Bril et al., 2024[60] | 204 participants were submitted to PNPLA3 genotyping, OGTT, MRS, and LB. A subgroup of 55 participants had an EHC with glucose tracer infusion | During MRA, A1c and Adipo-IR were associated with MASLD and advanced liver fibrosis, regardless of PNPLA3 genotype | Individuals with the PNPLA3 variant and MASLD were similarly insulin-resistant at various levels (liver, muscle, and AT) compared to non-variant carriers with MASLD |
| Elmansoury et al., 2024[61] | 205 MASLD cases and 187 healthy controls were recruited. Steatosis and fibrosis were assessed using Fibroscan | The PNPLA3 rs738409 C>G variant was linked to MASLD, fibrosis, steatosis, increased SBP and DBP, as well as elevated ALT (all P < 0.05) | This study shows that the PNPLA3 rs738409 C>G variant is associated with MASLD severity and BP among Egyptians |
| Kocas-Kilicarslan et al., 2024[62] | 123 patients with HC, 145 with MASH, and 72 cases of ESLD owing to MASLD were genotyped | The PNPLA3 rs738409: G was associated with the healthy state to MASH progression and from MASH to ESLD | The PNPLA3 alleles play a role in MASLD progression |
| Lavrado et al., 2024[4] | 407 patients with T2D-MASLD were followed for 11 years | Having ≥ 1 G or T allele of PNPLA3 was strongly linked to a higher risk of cirrhosis. The odds of complications from cirrhosis were higher in PNPLA3 GG (OR 27.20, 95%CI: | The PNPLA3 alleles are associated with the progression of MASLD to cirrhosis and its complications |
| Seko et al., 2024[63] | A longitudinal multicenter cohort study of 1,178 biopsy-proven MASLD cases | During MRA, PNPLA3 was found to be significantly associated with LREs (HR 1.91, 95%CI: 1.20-3.04) | Genetic variants predict LREs in MASLD among Japanese subjects |
| Pelusi et al., 2024[64] | A prospective study of 98 probands with advanced MASLD-fibrosis and/or MASLD-HCC, as well as 160 nontwin first-degree relatives who were assessed for MASLD at 4 referral centers in Italy 4 | Although the PNPLA3 risk variant was enriched in probands (P = 0.003) and over transmitted to relatives with MASLD (P = 0.045), evaluation of genetic risk variants and polygenic risk scores was not useful to direct noninvasive screening of advanced fibrosis among relatives | While these variants do play a role in liver disease within families, they do not improve the risk stratification of fibrosis |
| Suresh et al., 2024[65] | A retrospective survey conducted on 7,333 MASLD adults who were seen at the University of Michigan Health System. Out of this group, 1,468 individuals (20%) had elevated ferritin values | In a multivariable model, ferritinemia was linked to a higher mortality rate (HR 1.68, CI: 1.35-2.09, P < 0.001), incident LRE (HR 1.92, CI: 1.11-3.32, P = 0.019), and the PNPLA3-rs738409-G cirrhosis-promoting allele (P = 0.0068), but not to variants of the HFE gene | Metabolic hyperferritinemia is correlated with increased mortality and a higher likelihood of LRE, as well as cirrhosis-promoting alleles rather than HFE mutations that promote iron overload |
In contrast to previous studies conducted on polymorphisms of the Apolipoprotein B (apoB) gene[66], individuals with the PNPLA3 gene variant and MASLD are equally insulin-resistant at multiple levels: liver, muscle, and adipose tissue[60]. This insulin resistance likely drives the worsening of liver fibrosis in a mutual and bi-directional manner[67].
Of clinical interest, PNPLA3 gene variants interact with other gene variants, leading to increased severity of liver disease in cases of SERPINA 1[68], Apolipoprotein B[69,70], and α1 antitrypsin deficiency[71]. Conversely, HSD17B13 mitigates the effects of PNPLA3 on hepatic fibrosis[72,73]. All of these interactions should be carefully considered in the context of precision medicine approaches.
PNPLA3 gene variants affect extrahepatic outcomes in MASLD
Further to its liver-related effects, MASLD is a systemic condition typically associated with extrahepatic outcomes, including cardiovascular events, non-hepatic cancers, and chronic kidney disease (CKD)[2]. Additionally, MASLD may also affect the hepato-dermal axis, predisposing individuals to psoriasis[74]. Interestingly, PNPLA3 gene variants appear to modulate the entire spectrum of these extrahepatic manifestations [Table 4][75-82] through their effects on insulin resistance, lipid levels, and hepatic fibrosis. In this context, PNPLA3-I148M variants are associated with a decreased risk of coronary artery disease[76], an increased risk of extrahepatic cancers[81], and a detrimental effect on renal function in individuals with MASLD[79]. This renal effect seems to occur regardless of liver fibrosis[83].
Role of PNPLA3 gene variants and extrahepatic outcomes
| Author, year [Ref.] | Method | Findings | Conclusions |
| Karamfilova et al., 2019[75] | 208 MASLD individuals without (n = 125) and with pre-T2D (n = 83) were assessed | Compared to the wild CC genotype: - The CG genotype was associated with pre-T2D, IR, dyslipidemia and MetS - The PNPLA3-I148M variant exhibits a 9.6-fold higher odds of altered glucose metabolism - (OR 9.649, 95%CI: 2.100-44.328, P = 0.004) and a 3-fold higher odds of MetS (OR 2.939, 95%CI: 1.590-5.434, P = 0.001) and a 2.1-fold higher odds of IR (OR 2.127, 95%CI: 1.078-4.194, P = 0.029) | PNPLA3-I148M is associated with an increased risk of pre-T2D, MetS and IR among subjects with obesity and MASLD |
| Wu et al., 2020[76] | 189 subjects with MASLD and CAD, 242 individuals with MASLD and 242 HCs were enrolled | MASLD carriers of the CG + GG genotype exhibited a reduced CAD risk compared to those carrying the CC genotype (OR = 0.6, 95%CI: 0.40-0.90, | PNPLA3- I148M variants carry a decreased risk of CAD among MASLD subjects due to reduced lipidemic values |
| Ajmera et al., 2021[77] | A cross-sectional analysis of 264 middle-aged subjects submitted to genotyping and LSM with MRE. Advanced fibrosis was defined by liver stiffness | Each PNPLA3 risk variant copy carried an increase of 0.40 kPa (95%CI: 0.19-0.61, P < 0.01) in LS on MRE after adjusting for confounding factors. Moreover, a significant genotype-age interaction was found (P < 0.01) | The assessment of the PNPLA3 genotype may help identify individuals who, due to their high genetic risk, require closer monitoring and aggressive treatment |
| Moon et al., 2022[78] | A cross-sectional analysis of the Boromae MASLD cohort (n = 706) and a longitudinal cohort study of the GENIE study (n = 4,998) were conducted | Among MASLD subjects, the G allele was independently associated with a reduced risk of DM in both SLD (OR per 1 allele, 0.66, 95%CI: 0.46-0.97) and MASH (OR per 1 allele, 0.59, 95%CI: 0.38-0.92). This finding was confirmed by the longitudinal GENIE cohort The G allele was linked to a reduced odds of incident DM during the 60-month median follow-up in MASLD subjects (HR 0.65, 95%CI: 0.45-0.93). However, those carrying the G allele without NAFLD had higher risks of T2D (OR, 2.44, 95%CI: 1.00-5.95) in the Boromae cohort | This study supports the notion that “genetic MASLD” may have a lower risk of metabolic dysfunction |
| Mantovani et al., 2023[79] | A total of 1,144 middle-aged individuals were enrolled. The eGFR was determined using the CKD Epidemiology Collaboration equation. A subgroup of 144 cases were monitored for a median duration of 17 months | The p.I148M variant was associated with lower eGFRCKD-EPI levels | Among middle-aged subjects with metabolic dysfunction, the PNPLA3-I148M variant had a negative impact on renal function, regardless of common risk factors for CKD |
| Mantovani et al., 2023[80] | Outpatient cohort of 46 postmenopausal women with T2D and preserved kidney function at baseline followed for 5 years | During the 5-year follow-up, the rs738409 CG/GG genotypes were associated with faster eGFR decline (coefficient: -6.55, 95%CI: 11.0 to -2.08; P = 0.004 by random-effects panel data analysis) regardless of changes in confounding factors over 5 years | In postmenopausal T2D women, the risk allele (G) of PNPLA3 rs738409 is linked to a faster decline in eGFR during 5 years, regardless of yearly variations in common factors of risk of renal insufficiency and antidiabetic drugs |
| Tai et al., 2024[81] | Prospective Taiwanese cohort study of subjects with | A stratified analysis of the data showed that, among SLD subjects with the PNPLA3-I148M-rs738409 GG genotype (and also among those with the GC or CC genotype), the FIB-4 score was associated with incident non-hepatic cancers (HR 1.543, 95%CI: 1.195-1.993) | SLD individuals with the PNPLA3-I148M-rs738409 GG genotype who have high FIB-4 scores should be closely monitored for early diagnosis of emerging extrahepatic cancers |
| Agoglia et al., 2024[82] | A cross-sectional analysis of 199 prospectively enrolled subjects with psoriasis | T2D (OR 10.76, 95%CI: 2.42-47.87; P = 0.002) and carrying of ≥ 1 PNPLA3-G allele (OR 5.66, 95%CI: 1.08-29.52; P = 0.039) were linked to advanced liver fibrosis | Metabolic dysfunction and the PNPLA3-G allele, rather than TMSF2 variants and MTX therapy, are associated with fibrotic progression in MASLD |
Association of other lipid genes in the progression and severity of MASLD
Our understanding of gene-to-gene interactions is increasingly being elucidated, with well-characterized examples including 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) and transmembrane 6 superfamily 2 (TM6SF2). The HSD17B3 gene is located on human chromosome 4q22.1 and shows strong liver-specific expression[84]. Associated with lipid droplets, it catalyzes the interconversion between 17-keto and 17-hydroxysteroids, primarily contributing to liver-specific fatty acid metabolism involving lipid droplets. Additionally, it has retinol dehydrogenase (RDH) activity and acts as a binding protein for adipose triglyceride lipase (ATGL), facilitating the interaction between the comparative gene identification-58 (CGI-58), representing a 1-acylglycerol-3-phophate O-acyltransferase, and ATGL on hepatocyte lipid droplets[84]. The expression of HSD17B13 is induced by liver X receptor α via sterol regulatory element-binding protein 1c, which is a crucial transcription factor in the control of lipid metabolism. Importantly, overexpression of HSD17B13 is associated with increased ATGL- and RDH-mediated lipolysis, resulting in enhanced intrahepatic accumulation of lipid droplets. Conversely, a single nucleotide polymorphism (SNP) in HSD17B13 termed rs72613567:TA is associated with a prematurely truncated unstable protein with significantly reduced enzymatic activity[84].
Several missense variants of the TM6SF2 gene, such as E167K, L156P, and P216L, are also associated with higher odds of hepatic steatosis independent of the PNPLA3 I148M risk allele[85]. This gene is located on chromosome 19 (19p12) and acts as a crucial regulator of the hepatic homeostasis of lipids by influencing the secretion of triglycerides and the intra-hepatic content of lipid droplets[86]. The absence of the Tm6sf2 gene in mice is causally associated with SLD and raised liver enzymes independent of dietary challenge[87]. The study also revealed that TM6SF2 is required for normal lipidation of triglyceride-rich lipoproteins and is a key player in assembling very low density lipoprotein (VLDL), as evidenced by smaller VLDL particles with reduced triglyceride content. Moreover, disruption of Tm6sf2 resulted in significantly lowered Pnpla3 transcript levels, demonstrating that these genes are functionally linked to one another in regulating hepatic fat content[87].
In addition to variants in the HSD17B13 and TM6SF2 genes, there are other genetic variants that exert positive or negative metabolic effects in the pathogenesis of MASLD. These include, for example, the glucokinase regulatory protein (GCKR) variant rs1260326-T (P446L), the single nucleotide polymorphism (SNP) rs12137855-C within the lysophospholipase-like 1 (LYPLAL1) gene, and the variant rs641738C>T within the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) loci, suggesting that various molecular events contribute to the final outcome of MASLD[88,89]. Most genes associated with the initiation and progression of MASLD are driven either by excessive hepatic glucose levels leading to amplified lipogenesis (e.g., GCKR and LYPLAL1), reduced VLDL secretion (e.g., TM6SF2), or impaired triglyceride mobilization from hepatic lipid storage (e.g., PNPLA3) [Figure 4].
Figure 4. Representation of PNPLA3, HSD17B13, and TM6SF2 proteins in lipid metabolism and the progression of MASLD.
This graphical illustration summarizes how these three proteins interact in hepatocytes in relation to lipid metabolism. PNPLA3 is located on lipid droplets, HSD17B13 is found in the cytoplasm or endoplasmic reticulum (ER), and TM6SF2 is situated on the ER membrane. PNPLA3 catalyzes the hydrolysis of triglycerides into free fatty acids, HSD17B13 modules fatty acid, and steroid hormone metabolism to influence lipid homeostasis, while TM6SF2 facilitates the export of triglycerides from the liver into circulation. There are clear regulatory relationships among these proteins; changes in PNPLA3 activity could impact the size of lipid droplets (budding) and subsequently affect the ability of TM6SF2 to export lipids. Specific genetic variants or single nucleotide polymorphisms, such as PNPLA3-I148M or TM6SF2-E167K, TM6SF2-L156P, and TM6SF2-P216L, can influence protein activity, contributing to steatosis in MASLD progression. Additionally, the HSD17B13-rs72613567:TA is associated with a prematurely truncated unstable HSD17B13 protein with significantly reduced enzymatic activity.
Moreover, a recent meta-analysis that analyzed 399 eligible studies identified 11 variants in 10 genes that were significantly associated with MASLD, with cumulative epidemiological evidence. The association was graded strong for the homeostatic iron regulator (HFE) and the tumor necrosis factor (TNF) genes, moderate for four variants located either in TM6SF6, GCKR, or the adipose most abundant gene transcript-1 (ADIPOQ), and weak for five variants located in MBOAT7, phosphatidylethanolamine N-methyltransferase (PEMT), PNPLA3, leptin receptor (LEPR), and methylenetetrahydrofolate reductase (MTHFR)[90]. For example, PNPLA3 and TM6SF2 E167K variants are associated with an increased susceptibility to MASLD development and progression, while HSD17B13 may provide protection against these liver-related outcomes[91]. This suggests that different gene variants could have reinforcing or mitigating effects on pathogenesis, complicating outcome predictions. Therefore, polygenic risk scores are calculated by summing the number of risk alleles (e.g., PNPLA3, TM6SF2) and subtracting the protective variant found in HSD17B13[92].
However, some genetic variants and SNPs may not be associated with the MASLD risk in certain populations or may not significantly impact mortality between lean and non-lean populations with MASLD[93,94]. This indicates that specific gene polymorphisms affecting MASLD development can vary significantly across populations due to genetic, environmental, and lifestyle factors. Variants that pose a risk or offer protection in one population may not have the same effect in another due to differences in allele frequencies, gene-environment interactions, and cultural practices influencing diet and physical activity. It is important to note that the benefits of dietary interventions and probiotics, which are reasonable strategies for managing MASLD, may differ among genotype groups, such as individuals with different PNPLA3 genotypes[95]. Understanding these population-specific effects and the impact of genotype constellations is crucial for developing tailored prevention strategies and personalized medical interventions that account for genetic diversity.
PNPLA3 gene variants in people living with HIV
Among people living with HIV (PLWH), hepatic disorders account for high morbidity and liver-related causes are among the leading causes of death not related to acquired immunodeficiency syndrome (AIDS)[96]. SLD is common among PLWH[97], including those with normal BMI, in whom it is generally deemed to be associated with antiretroviral treatment (ART)[98]. Moreover, a previous study has indicated that HIV is a steatogenic virus[99].
Based on these findings, it is predicted that mono-infection with HIV amplifies the steatogenic potential of variants of the PNPLA3 gene. A recent study by Han et al. seemingly confirms this prediction[100]. These authors cross-sectionally investigated PNPLA3 variants and either SLD or MASLD in a Thai patient sample of 764 PLWH, 35% of whom had SLD. In multivariate analysis adjusted for common confounding factors, including ART, PNPLA3 rs738409 CG/GG genotypes were found to be associated with higher odds of SLD only among lean subjects (aOR: 1.79, 95%CI: 1.18-2.72, P = 0.006). This finding remained significant after adjustment for TM6SF2 rs5854292 CT/TT and CC genotypes (2.01, 95%CI: 1.24-3.25, P = 0.004) in lean participants[100]. These findings support the notion that PNPLA3 gene variants may be an independent contributor to MASLD development, suggesting longitudinal follow-up of these subjects[101].
PNPLA3 gene variants in the liver transplant setting
MASH-cirrhosis is the most rapidly growing indication for liver transplantation in the Western world[102]. Metabolic factors increase the likelihood of mortality among those on the waiting list for MASH-cirrhosis and the risks of long-term recurrence of liver disease and cardiometabolic complications after liver transplantation[103]. A recent retrospective study of 55 Japanese SLD recipients and their donors found that donor risk alleles of PNPLA3, TM6SF2, and HSD17B13 are implicated in post-transplant SLD, rather than recipient risk alleles[104]. Another study of 83 liver recipients showed that PNPLA3 gene variants in the recipient genotype impact the post-transplant outcome of individuals transplanted for alcohol-related liver disease, especially in those with heavy alcohol relapse[105].
PNPLA3 gene variants in children and adolescents with MASLD
In contrast to adults, MASLD in younger age groups is not expected to be associated with clinically manifest cardiovascular disease or cancer. Nevertheless, data from two meta-analytic reviews suggest that PNPLA3 variants participate in the development and fibrotic progression of MASLD among children and adolescents[106,107]. Tang et al. conducted a study that included nine case-control studies totaling 1,173 children with MASLD and 1,792 healthy controls[106]. The data have demonstrated that PNPLA3 gene variants were strongly associated with the risk of development and progression of NAFLD in children.
Ethnicity, dietary habits, obesity, and PNPLA3 gene variants are likely to interact in the initiation and worsening of MASLD among children and adolescents. In agreement, Mansoor[108] found that, among Hispanic children with obesity, the PNPLA3 rs738409 C>G polymorphism was associated with an increased risk of MASLD. Schenker et al. have reported that, in Latino adolescents with obesity, the PNPLA3 gene GG variant, total sugar, fructose, sucrose, and glucose consumption were all associated with liver stiffness measurement, a noninvasive index of liver fibrosis, in a stronger manner among cases with fibrosing MASLD than among healthy controls and MASLD without fibrosis[109]. Gene-to-gene interactions between the PNPLA3 gene and other gene variants (such as TM6SF2 and SAMM5) concur in the development and progression of MASLD in children[110]. This finding, together with the observation that children with obesity and PNPLA3-MM genotype compared to other genotypes, exhibit reduced renal function, particularly if MASLD coexists[111], pave the way for identifying specific subsets of individuals who, being exposed to the risks of worse outcomes, need more aggressive treatment strategies.
PNPLA3 and autoimmune hepatitis
The PNPLA3 rs738409 GG variant is associated with prognostic features of AIH, such as time to liver transplantation or death[112]. More recently, Azariadis et al. conducted a study involving 200 AIH cases and 100 healthy controls and found that the I148 M variant was present in the same proportion among AIH patients as in healthy controls (47.5% vs. 47%, P = 1.000)[113]. However, AIH subjects with the GG/CG genotypes were associated with decompensated cirrhosis at diagnosis (GG/CG 6.3% vs. CC 1%,
Utility of PNPLA3 in clinical practice
The incorporation of PNPLA3 genotyping in clinical practice remains challenging. The European Clinical Practice Guidelines on MASLD management that were recently published clearly state that genotyping should only be performed in the clinical research setting[2]. However, Chen and Vespasiani-Gentilucci have proposed a hierarchy of potential relevance, which is summarized in Table 5[115].
Potential integration of PNPLA3 genotyping into clinical risk prediction1
| Hierarchy | Indication | Comment |
| More relevant | Diagnosis of steatohepatitis | This is best accomplished noninvasively with FAST and MAST scores. However, the role of PNPLA3 genotyping and PRS remains to be determined |
| Less relevant | Risk stratification of non-cirrhotic MASLD | The inclusion of PNPLA3 genotyping in clinical practice would be facilitated by demonstrating that the genotype is associated with LROs independent of established clinical risk scores in both the general population and among those with MASLD |
| Risk stratification among those with cirrhosis | Severe LROs include decompensation of cirrhosis and the development of HCC | |
| Diagnosis of SLD | Various NITs accurately identify SLD, and the addition of PNPLA3 genotyping minimally improves their diagnostic accuracy | |
| Staging of fibrosis | Liver histology remains the reference standard and NITs accurately predict liver histology findings |
Although well documented, the ranking illustrated in Table 5 remains “Expert Opinion”. While specialized centers may consider incorporating the assessment of the genetic risk profiles (comprising PNPLA3 p.I148M variant and/or polygenic risk scores) to personalize risk stratification, this practice has yet to be validated with large, prospective studies[2].
Relationship between PNPLA3, alcohol-related liver disease, and metabolic and alcohol-related/associated liver disease (MetALD)
A seminal meta-analytic review, pooling data from 10 published studies globally involving 4,112 individuals, has reported several important findings on the connection between PNPLA3 gene variants and alcohol-related liver disease (ALD)[116]. According to this study, the OR for rs738409 CG and GG among alcohol-related cirrhosis (AC) patients was 2.09 (1.79-2.44) and 3.37 (2.49-4.58), respectively vs. controls. Among AC patients with HCC, the OR was 2.87 (1.61-5.10) for CG and 12.41 (6.99-22.03) for GG. For ALD patients, the OR of CG and GG genotypes was 2.62 (1.73-3.97) and 8.45 (2.52-28.37), respectively, for AC compared with SLD subjects. The OR for CG and GG genotypes among AC patients for HCC occurrence was 1.43 (0.76-2.72) and 2.81 (1.57-5.01), respectively. These findings collectively support the idea that, among drinkers, the PNPLA3 rs738409 polymorphism is associated with higher odds for the whole ALD spectrum and a higher likelihood of developing AC and HCC[117].
In the Delphi consensus conducted by Rinella et al. in 2023, a new category named MetALD was introduced outside of pure MASLD - MetALD is used to identify individuals with MASLD who also consume 140-350 grams of alcohol weekly for females and 210-420 grams of alcohol weekly for males[116]. Due to the recent introduction of the MetALD nomenclature, we still ignore the existence and the extent of the expected interaction of PNPLA3 gene variations with MetALD. However, the contribution of PNPLA3 I148M to the global burden of MetALD may vary depending on the prevalence of dysmetabolic traits among different world regions[6].
CONCLUSION
The spectrum of PNPLA3-driven liver disease represents an extraordinary naturally occurring disease model that can be utilized to better understand how precision medicine approaches may be implemented in human medicine. However, it is true that this expectation is still far from reality, although some firm conclusions are at hand.
Regarding diagnostics, clinicians and researchers should consider that the PNPLA3 genotype reduces the accuracy of noninvasive assessment of MASLD[118]. Additionally, no evidence is presently available to support the use of genetic risk scores for identifying significant fibrosis in MASLD, although the combined use of PNPLA3 and Fib-4 considerably increases diagnostic accuracy[119].
When it comes to disease stratification, having the PNPLA3-I148M variant can increase the odds of the more severe forms of MASLD, particularly in women[120]. This variant could also play a role in pharmacogenetics[121]. It is likely that all types of interventions, whether lifestyle changes or medication, for MASLD are influenced by the PNPLA3 genotype. Therefore, considering the PNPLA3-I148M variant status in therapeutic studies could help prevent inaccurate results due to this potentially confounding factor[122].
Profiling PNPLA3 variants is of significant value in the field of treatment. Currently, no drug has been approved to target the PNPLA3-I148M variant specifically, but precision medicine approaches in this area are anticipated[123]. In elderly Japanese individuals at risk of MASLD, the PNPLA3 rs738409 genotype may be linked to the positive effects of physical exercise[124]. Another potential option could be vitamin B3 supplementation, as there is an interaction between niacin and PNPLA3 I148M in MASLD patients[125]. Gene silencing appears to be the most direct approach[126]. However, a recent two-sample, two-step Mendelian randomization analysis investigating the relationship between PNPLA3 inhibition and cardiovascular diseases (CVDs) found that inhibiting PNPLA3 gene expression increases the risk of major CVDs[127]. A seminal investigation has suggested that PNPLA3(148M) is a gain-of-function mutation that promotes hepatic steatosis by accumulating on LDs and inhibiting ATGL-mediated lipolysis in an ABHD5-dependent manner[128]. Based on these findings, it is anticipated that reducing (as opposed to increasing) the expression of PNPLA3 would be the most successful strategy to treat PNPLA3(148M)-associated SLD. Collectively, these conflicting results highlight the liver’s role as a reservoir of lipid species and a complex regulator of cardiovascular risk. They suggest the need for additional studies following a more holistic, sex-specific, and well-balanced approach to managing hepatic and extrahepatic outcomes simultaneously.
DECLARATIONS
Authors’ contributions
Made substantial contributions to conception and design of the review: Weiskirchen R, Lonardo A
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Financial support and sponsorship
None.
Conflicts of interest
Weiskirchen R is Associate Editors of the Journal of Translational Genetics and Genomics and is the Guest Editors for the special issue Genetic and Epigenetic Factors in Liver Disease Pathogenesis. Weiskirchen R was not involved in any steps of edtorial processing, notably including reviewers' selection, manuscript handling and decision making. while the other author have declared that they have no conflicts of interest.
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Copyright
© The Author(s) 2024.
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, Amedeo LonardoHow to Cite
Weiskirchen, R.; Lonardo, A. PNPLA3 as a driver of steatotic liver disease: navigating from pathobiology to the clinics via epidemiology. J. Transl. Genet. Genom. 2024, 8, 355-77. http://dx.doi.org/10.20517/jtgg.2024.70
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