International Journal of Molecular Sciences Article Impact of a Loss-of-Function Variant in HSD17B13 on Hepatic Decompensation and Mortality in Cirrhotic Patients Antonio Gil-Gómez 1,2 , Ángela Rojas 1,2 , María R. García-Lozano 1,2 , Rocío Muñoz-Hernández 1,2 , Rocío Gallego-Durán 1,2 , Douglas Maya-Miles 1,2 , Rocío Montero-Vallejo 1,2 , Sheila Gato 1,2 , Javier Gallego 1 , Rubén Francés 2,3,4 , Germán Soriano 2,5 , Javier Ampuero 1,2,6 and Manuel Romero-Gómez 1,2,6, * 1 SeLiver Group, Institute of Biomedicine of Seville (IBiS), Virgen del Rocio University Hospital, CSIC, University of Seville, 41013 Seville, Spain 2 CIBERehd, Instituto de Salud Carlos III, 28029 Madrid, Spain 3 Clinical Medicine Department and IDIBE, Miguel Hernández University, 03550 San Juan de Alicante, Spain 4 IIS ISABIAL, Hospital General Universitario de Alicante, 03010 Alicante, Spain 5 Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain 6 UCM Digestive Diseases, Virgen del Rocío University Hospital, 41013 Seville, Spain * Correspondence: mromerogomez@us.es Abstract: A common splice variant in HSD17B13 (rs72613567:TA) was recently found to be associated with a reduced risk of developing chronic liver disease in NAFLD patients and a reduced risk of progression to advanced fibrosis and cirrhosis. In this study, we aimed to evaluate the prognosis of cirrhotic patients harboring this variant. We performed a retrospective analysis on 483 prospectively Citation: Gil-Gómez, A.; Rojas, Á.; recruited patients from four different hospitals in Spain, followed-up for at least 5 years. We collected García-Lozano, M.R.; Muñoz- clinical, demographic, and biochemical data, and we performed a genotyping analysis for com- Hernández, R.; Gallego-Durán, R.; mon variants previously associated with liver disease risk (HSD17B13 rs72613567:TA and PNPLA3 Maya-Miles, D.; Montero-Vallejo, R.; rs738409). Patients homozygous for the TA allele showed a higher MELD score (p = 0.047), Child– Gato, S.; Gallego, J.; Francés, R.; et al. Impact of a Loss-of-Function Variant Turcotte–Pugh score (p = 0.014), and INR levels (p = 0.046), as well as decreased albumin (p = 0.004) at in HSD17B13 on Hepatic baseline. After multivariate analysis, patients with the “protective” variant indeed had an increased Decompensation and Mortality in risk of hepatic decompensation [aHR 2.37 (1.09–5.06); p = 0.029] and liver-related mortality [aHR 2.32 Cirrhotic Patients. Int. J. Mol. Sci. (1.20–4.46); p = 0.012]. Specifically, these patients had an increased risk of developing ascites (Log-R 2022, 23, 11840. https://doi.org/ 11.6; p < 0.001), hepatic encephalopathy (Log-R 10.2; p < 0.01), and higher mortality (Log-R 14.1; 10.3390/ijms231911840 p < 0.001) at 5 years of follow-up. Interactions with the etiology of the cirrhosis and with the variant Academic Editors: Stefania rs738409 in PNPLA3 are also described. These findings suggest that the variant rs72613567:TA in Grimaudo and Rosaria HSD17B13 has no protective effect, but indeed increases the risk of decompensation and death in Maria Pipitone patients with advanced chronic liver disease. Received: 7 September 2022 Keywords: cirrhosis; polymorphism; PNPLA3; HSD17B13; NAFLD; fibrosis; ascites; hepatic Accepted: 4 October 2022 encephalopathy; SNP; hepatic decompensation Published: 6 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Chronic liver disease progresses from fibrosis to cirrhosis, and ultimately to hepatic failure and hepatocellular carcinoma (HCC), being prominent causes of illness and death globally. The most common etiologies of cirrhosis are alcohol consumption, chronic hepati- Copyright: © 2022 by the authors. tis C or B infection, and non-alcoholic fatty liver disease (NAFLD), whose prevalence is Licensee MDPI, Basel, Switzerland. increasing in parallel with that of metabolic syndrome [1,2]. Cirrhosis progression results This article is an open access article in portal hypertension and hepatic decompensation, both of which substantially increase distributed under the terms and the risk of mortality, accounting for approximately 2 million deaths worldwide [3]. No- conditions of the Creative Commons tably, liver disease progression shows considerable interindividual variability; it has been Attribution (CC BY) license (https:// estimated that host genetic factors could explain approximately half of this variability, creativecommons.org/licenses/by/ contributing to the progression of cirrhosis and predisposing patients to the development 4.0/). of liver-related events [4,5]. Int. J. Mol. Sci. 2022, 23, 11840. https://doi.org/10.3390/ijms231911840 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 11840 2 of 11 Genome-wide association studies have identified sequence variations with robust effects on multiple stages of the disorder. The most validated so far is the variant p.I148M in 1-acylglycerol-3-phosphate O-acyltransferase (PNPLA3), associated with higher triglyc- eride levels, as well as with an increased risk of non-alcoholic steatohepatitis, cirrhosis decompensation, HCC development, and mortality [6–9]. Recently, an indel variant (T > TA variant, rs72613567) in the 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) gene, leading to the production of a truncated protein with reduced activity, has been associated with decreased aminotransferase levels and a reduction of liver injury in the setting of a fatty liver, mitigating the risk conferred by the carriage of the PNPLA3 variant [10,11]. Additionally, this variant has been associated with lower odds of developing cirrhosis and HCC among alcohol misusers [12,13]. However, the physiological role of this protein and the mechanisms underlying these associations have yet to be elucidated, since much of the genetic risk of chronic liver disease remains unexplained. The goal of this study was to evaluate the clinical impact of the genetic variant rs72613567:TA in HSD17B13 on the prognosis of cirrhotic patients and to explore possible risk interactions with the underlying etiology or the genotype of PNPLA3 rs738409. 2. Results 2.1. Baseline Characteristics and Genotyping The clinical and demographic characteristics of the patients are shown in Table 1. The majority of the patients were male (68.7%), with a mean age of 59.4 ± 11.9 years old. The most frequent etiology was alcohol (48.7%), followed by viral hepatitis (35.8% C and 6.2% B), with the other 9.3% being NAFLD, primary biliary cirrhosis, or autoimmune- related cirrhosis. At the time of inclusion, liver function was CTP score A, with a Model for End-stage Liver Disease (MELD) score of 12.2 ± 5.1, in 48.9% of patients. Table 1. Comparison of clinical and demographic characteristics of the patients according to the genotype of HSD17B13 rs72613567. HSD17B13 Total TA/TA Variable T/T or T/TA p-Value (n = 483) (n = 56) (n = 427) Age ± SD, years 59.4 ± 11.9 59.2 ± 11.5 60.8 ± 14.8 0.448 Male sex 68.7% (332/483) 68.1% (291/427) 73.2% (41/56) 0.442 CTP 7.0 ± 1.9 6.9 ± 1.9 7.6 ± 1.8 0.014 Etiology Alcohol 48.7% (235/483) 49.2% (210/427) 44.6% (25/56) 0.800 Viral 42.0% (203/483) 41.7% (178/427) 44.6% (25/56) Other 9.3% (45/483) 9.1% (39/427) 10.7% (6/56) MELD ± SD 12.2 ± 5.1 12.0 ± 5.1 13.4 ± 5.0 0.047 Albumin ± SD (g/L) 35.0 ± 7.9 35.4 ± 7.8 32.2 ± 7.6 0.004 Bilirubin ± SD (mg/dL) 2.14 ± 2.17 2.13 ± 2.14 2.24 ± 2.46 0.727 Platelets ± SD (×109 /L) 121.3 ± 70.0 120.8 ± 64.9 133.1 ± 100.9 0.340 Creatinine ± SD (mg/dL) 0.98 ± 0.45 0.98 ± 0.46 0.96 ± 0.36 0.762 AST ± SD (IU/L) 60.5 ± 57.8 61.5 ± 60.4 53.3 ± 31.9 0.315 ALT ± SD (IU/L) 46.0 ± 60.5 47.3 ± 63.9 36.0 ± 20.6 0.188 INR ± SD 1.32 ± 0.32 1.32 ± 0.32 1.40 ± 0.32 0.046 Leukocytes ± SD (cells/×109 /L) 6.96 ± 5.99 7.00 ± 6.19 6.63 ± 4.12 0.681 For the genotyping of rs72613567, we determined that 59.8% of patients were homozy- gous for the T allele, with 11.6% harboring the TA/TA variant (Figure 1A), resulting in a MAF (TA allele) of 0.255, comparable with that of previous studies (Abul-Husn et al., MAF = 0.242 [10]; Scheiner et al., MAF = 0.219) [14]. Nonetheless, we observed a deviation from the Hardy–Weinberg equilibrium (p < 0.05) for the variant in this cohort, which could indicate that the risk of disease per se is influenced by the polymorphism. For the genotyping of rs72613567, we determined that 59.8% of patients were homo- zygous for the T allele, with 11.6% harboring the TA/TA variant (Figure 1A), resulting in a MAF (TA allele) of 0.255, comparable with that of previous studies (Abul-Husn et al., MAF = 0.242 [10]; Scheiner et al., MAF = 0.219) [14]. Nonetheless, we observed a deviation Int. J. Mol. Sci. 2022, 23, 11840 3 of 11 from the Hardy–Weinberg equilibrium (p < 0.05) for the variant in this cohort, which could indicate that the risk of disease per se is influenced by the polymorphism. (n=483) (gnomAD n=55.616) A B 80 p = 0.008 60 T/T Frequency T/T (59.83%) T/TA or TA/TA 40 T/TA (28.57%) 20 TA/TA (11.59%) 0 n s Total=483 nt tio tie la pu pa po tic ho an irr pe C ro Eu C D ALT p = 0.001 HSD17B13 genotype 60 80 p = 0.335 HSD17B13 genotype T/T or T/TA T/T 60 TA/TA % of patients 50 T/TA U/L TA/TA 40 40 20 30 0 T/T T/TA TA/TA Child-Pugh A Child-Pugh B/C Figure 1. Figure (A) Genotype 1. (A) Genotype frequency frequency for for rs72613567. rs72613567. (B) (B) Comparison Comparison ofof minor minor allele allele frequency frequency (MAF) (MAF) across studies and in regards to liver function. (C) Alanine aminotransferase levels in patients with different genotypes genotypes (ANOVA). (ANOVA).(D)(D)Distribution Distributionofofgenotypes genotypesaccording accordingto to liver liver function function measured measured by by CTPCTP score. score. Compared to toaacontrol controlEuropean European population population (publicly available (publicly from from available https://gnomAD. https://gno- broadinsitute.org (accessed mAD.broadinsitute.org on 22 on (accessed September 22 September n = 55,616), 2022);2022); as in [13], n = 55,616), as inthe frequency [13], the fre- of TA allele quency of TA carriers was lower allele carriers wasinlower cirrhotic patientspatients in cirrhotic (40.2% vs. vs. p46.2%; 46.2%; (40.2% = 0.008), p = as shown 0.008), as in Figure 1B. This suggests, in agreement with previous reports, a protective shown in Figure 1B. This suggests, in agreement with previous reports, a protective effect effect of the variant of againstagainst the variant cirrhosis compared cirrhosis to healthy compared subjects.subjects. to healthy This effect was This maintained effect for ALD was maintained (OR: 0.68; CI 95% 0.53–0.86; p = 0.001), but not for viral etiology (OR: 0.95; for ALD (OR: 0.68; CI 95% 0.53–0.86; p = 0.001), but not for viral etiology (OR: 0.95; 95% 95% CI, 0.72–1.25; p = 0.72-1.25; CI, 0.725). In paddition, = 0.725).we In observed addition, the expected trend we observed towardstrend the expected decreased towardsALT levels in decreased patients ALT with levels in the TA allele patients with(Figure the TA1C).allele (Figure 1C). Curiously, the Curiously, the frequency frequency of of patients patients with with worse worse liver liver function function (CTP (CTP score score B/C) B/C) waswas higher among those carrying the TA/TA genotype (Figure 1D). higher among those carrying the TA/TA genotype (Figure 1D). Furthermore, we alsoFurthermore, we also found that MELD found that MELD (12.0 ±(12.0 scores scores 5.1 vs. 13.4 ±5.1 vs.±13.4 p 5.0; 5.0; ± < 0.05) p < and 0.05)International Normalized and International Ratio Normalized (INR) levels (1.32 ± 0.32 vs. 1.40 ± 0.32; p < 0.05) were increased and albumin Ratio (INR) levels (1.32 ±0.32 vs. 1.40 ±0.32; p < 0.05) were increased and albumin levels levels were decreased (35.4 ± 7.8 vs. 32.3 ± 7.6; p < 0.05) in rs72613567 TA/TA patients were decreased (35.4 ±7.8 vs. 32.3 ±7.6; p < 0.05) in rs72613567 TA/TA patients (Table 1). (Table 1). 2.2. Impact of rs72613567 Genotype on Mortality and Hepatic Decompensation 2.2. Impact of rs72613567 Genotype on Mortality and Hepatic Decompensation The patients were followed for a median of 36.8 ± 23.9 months. During that period, The patients were followed for a median of 36.8 ±23.9 months. During that period, 47 patients (9.7%) underwent liver transplantation. Of the remaining patients, 111 (25.5%) 47 patients (9.7%) underwent liver transplantation. Of the remaining patients, 111 (25.5%) died within five years, 92 of which (82.3% of the deaths) were related to their underlying died within five years, 92 of which (82.3% of the deaths) were related to their underlying liver disease. Surprisingly, carriers of the TA/TA genotype not only were not protected, liver disease. Surprisingly, carriers of the TA/TA genotype not only were not protected, but indeed had an increased risk of liver transplantation [HR 1.56 (95% CI 0.70–3.50); but indeed haddeath p = 0.275] and an increased risk of liver [1.96 (1.22–3.15); p= transplantation [HRif1.56 0.005], especially (95%into taking CI 0.70–3.50); p= account liver- related deaths [2.52 (1.53–4.14); p = 0.001]. The cumulative incidence of liver-related death or transplantation is shown in Figure 2A. The resulting regression models, after adjusting for age, sex, liver function (MELD score), and factors influencing the progression of cirrhosis (i.e., alcohol intake or active viral infection), confirmed this trend (Table 2). 0.275] and death [1.96 (1.22–3.15); p = 0.005], especially if taking into account liver-related deaths [2.52 (1.53–4.14); p = 0.001]. The cumulative incidence of liver-related death or transplantation is shown in Figure 2A. The resulting regression models, after adjusting Int. J. Mol. Sci. 2022, 23, 11840 for age, sex, liver function (MELD score), and factors influencing the progression of4 cir- of 11 rhosis (i.e., alcohol intake or active viral infection), confirmed this trend (Table 2). p = 0.079 A B 100 100 Liver transplantation rs72613567 T/T-T/TA Liver transplantation rs72613567 TA/TA N° of complications Liver-related death rs72613567 T/T-T/TA 17.9 log-Rank=14.1 29.6 5 % of patients Liver-related death rs72613567 TA/TA Probability 4 50 50 26.7 3 2 35.2 1 24.3 0 20.4 20.3 9.3 0 0 0 12 24 36 48 60 T/T or T/TA TA/TA Time to liver transplantation or liver-related death (months) C D 100 100 Ascites rs72613567 T/T-T/TA HE rs72613567 T/T-T/TA log-Rank=11.6 log-Rank=10.2 Ascites rs72613567 TA/TA HE rs72613567 TA/TA Ascites development (%) Hepatic encephalopathy development (%) 50 50 0 0 0 12 24 36 48 60 0 12 24 36 48 60 Time (months) Time (months) Figure 2. Figure (A) Mortality 2. (A) Mortality with with liver liver transplantation transplantation as as aa competing competing event. event. (B) (B) Frequency Frequency ofof patients patients with an with anincreasing increasingnumber numberofofcirrhosis cirrhosis complications. complications. Cumulative Cumulative incidence incidence of ascites of (C) (C) ascites and and (D) (D) hepatic hepatic encephalopathy encephalopathy development, development, according according to the the HSD17B13 to HSD17B13 rs72613567 rs72613567 genotype. genotype. Table Table 2.2. Multivariate MultivariateCox Cox regression regression analyses analyses onimpact on the the impact of the genotype of the genotype of HSD17B13 of HSD17B13 rs72613567 rs72613567 on liver transplantation, mortality, and hepatic decompensation. on liver transplantation, mortality, and hepatic decompensation. Any (Fur- Any (Further) Liver Transplantation Mortality Liver-Related Death Liver Transplantation Mortality Liver-Related Death ther)$$$$$$Decom- Decompensation aHR aHR aHR aHRpensation p-Value p-Value p-Value p-Value (95% CI) aHR$$$$$$(95 (95% CI) (95% CI) aHR$$$$$$(95 aHR$$$$$$(95 (95% CI) aHR$$$$$$(95 1.96 % CI) p-Value 1.17 p-Value 1.27 p-Value 1.04 p-Value Sex 0.097 % CI) 0.531 % CI) 0.404 % CI) 0.880 (0.87–4.32) (0.71–1.93) (0.72–2.21) (0.64–1.70) 1.96$$$$$$(0.8 0.98 1.17$$$$$$(0.7 1.05 1.27$$$$$$(0.7 1.05 1.04$$$$$$(0.6 1.04 AgeSex 0.089 0.097 0.0010.531 0.404 (1.02–1.06) 0.001 0.880 0.001 (0.95–1.00) 7–4.32) (1.03–1.07) 1–1.93) (1.03–1.08) 2–2.21) 4–1.70) 1.09 1.09 1.11 1.29 MELD 0.98$$$$$$(0.9 (1.04–1.15) 0.001 1.05$$$$$$(1.0 0.001 1.05$$$$$$(1.00.001 1.04$$$$$$(1.0 0.001 Age 0.089(1.05–1.14) 0.001 (1.07–1.16) 0.001 (1.21–1.38) 0.001 1.52 5–1.00) 1.523–1.07) 3–1.08) 1.39 2–1.06) 1.99 Etiological factors 0.197 0.069 0.204 0.002 (0.81–2.84) (0.97–2.39) (0.84–2.30) (1.29–3.07) 1.09$$$$$$(1.0 1.09$$$$$$(1.0 1.11$$$$$$(1.0 1.29$$$$$$(1.2 rs72613567 MELDTA/TA 1.21 0.001 1.67 0.001 2.32 0.001 2.37 0.001 4–1.15) 0.673 5–1.14) 0.089 7–1.16) 0.012 1–1.38) 0.029 genotype (0.49–2.93) (0.91–3.18) (1.20–4.46) (1.09–5.06) 1.52$$$$$$(0.8 1.52$$$$$$(0.9 MELD: Model for End-Stage Liver Disease; etiological1.39$$$$$$(0.8 1.99$$$$$$(1.2 factors: relapse in alcohol intake or active viral infection. Etiological factors 0.197 0.069 0.204 0.002 1–2.84) 7–2.39) 4–2.30) 9–3.07) rs72613567 TA/TA geno- 1.21$$$$$$(0.4 In terms of decompensation, 1.67$$$$$$(0.9 patients with the variant in HSD17B13 2.32$$$$$$(1.2 had an increased 2.37$$$$$$(1.0 cumulative 0.673 of complications0.089 number of cirrhosis (Figure 2B). 0.012 Specifically, 0.029 type 9–2.93) 1–3.18) 0–4.46) 9–5.06) patients had these a higher MELD: risk of Model fordeveloping End-Stage Liverascites (log-Rank Disease; etiological p < 0.001) 11.6; factors: and in relapse hepatic alcoholencephalopathy intake or active (log-Rank 10.2; viral infection. p < 0.001) (Figure 2C,D). The Kaplan–Meier curves for the cumulative incidence of other complications, such as SBP (p = 0.059), EVB (p = 0.427), or hepatorenal syndrome (p = 0.236), did not differ significantly, probably due to the reduced number of cases in the cohort. Furthermore, and in contrast to recent studies, carriers of the TA/TA genotype were not protected from the 5-year development of HCC (log-Rank 0.1; p = 0.801). 2.3. Effect of Etiology and Interactions with PNPLA3 Genotype Previous studies have adjudged HSD17B13 to have a role in lipid droplet metabolism, and, so far, most of the literature shows associations with non-viral liver disease (ALD/NAFLD Int. J. Mol. Sci. 2022, 23, 11840 5 of 11 patients). We wondered if the underlying etiology of cirrhosis could differentially affect the prognosis of patients harboring the variant rs72613567. In order to find out, we compared patients with ALD/NAFLD to those with viral etiology. Of those with steatohepatitis, only 8.2% (21/256) had NAFLD; viral etiology was comprised 148 (72.9%) patients with hepatitis C virus (HCV) and 55 (27.1%) with hepatitis B virus (HBV). As shown in Table 3, patients with viral hepatitis carrying the variant had worse liver function, measured by either Child–Turcotte–Pugh (CTP) (7.8 ± 1.7 vs. 6.9 ± 2.0; p = 0.038) or MELD (13.4 ± 4.3 vs. 11.4 ± 4.8; p = 0.065), at baseline than those with the major allele (T-). In addition, INR levels (p = 0.009) were higher, and albumin levels were decreased (p = 0.001). Although the trend was similar, there were no significant changes in baseline clinical and biochemical parameters among ALD/NAFLD patients, suggesting that the genotype may influence differently depending on the etiology. Table 3. Comparison of patients’ characteristics according to the genotype of HSD17B13, depending on the etiology of the cirrhosis. ALD/NAFLD (n = 256) Viral Hepatitis (n = 203) Variable HSD17B13 HSD17B13 p-Value p-Value T/T or T/TA TA/TA T/T or T/TA TA/TA Age ± SD, years 59.1 ± 10.1 58.8 ± 13.2 0.853 59.3 ± 13.2 63.2 ± 16.4 0.259 Male sex 76.9% (176/229) 81.4% (22/27) 0.316 59.0% (105/178) 64.0% (16/25) 0.442 CTP 6.8 ± 1.9 7.4 ± 1.9 0.167 6.9 ± 2.0 7.8 ± 1.7 0.038 MELD ± SD 12.3 ± 5.2 13.8 ± 5.6 0.236 11.4 ± 4.8 13.2 ± 4.3 0.065 Albumin ± SD (g/L) 35.8 ± 7.7 34.1 ± 8.4 0.309 35.1 ± 8.1 29.7 ± 6.2 0.001 Bilirubin ± SD 2.2 ± 2.37 2.18 ± 1.90 0.816 1.94 ± 1.73 2.33 ± 3.05 0.346 (mg/dL) Platelets ± SD 125.3 ± 65.4 141.1 ± 69.1 0.279 112.1 ± 63.5 125.9 ± 131.5 0.610 (×109 /L) Creatinine ± SD 0.98 ± 0.49 0.98 ± 0.42 0.739 0.95 ± 0.40 0.95 ± 0.25 0.938 (mg/dL) AST ± SD (IU/L) 54.7 ± 63.9 48.5 ± 29.4 0.620 71.3 ± 56.3 59.5 ± 34.5 0.121 ALT ± SD (IU/L) 36.1 ± 42.5 31.8 ± 19.1 0.585 63.3 ± 83.5 41.2 ± 21.9 0.191 INR ± SD 1.34 ± 0.34 1.39 ± 0.31 0.555 1.27 ± 0.27 1.44 ± 0.32 0.009 Leukocytes ± SD 7.11 ± 4.58 8.19 ± 4.87 0.328 6.93 ± 8.18 5.10 ± 2.64 0.293 (cells/×109 /L) The etiology seemed to influence the cumulative incidence of cirrhosis complications. Patients with viral hepatitis carrying the mutation had an increased risk of developing any complication at five years (87.0% vs. 57.4%; log-R 9.8; p = 0.002) than those with the major allele, but we did not observe a significant difference among patients with ALD/NAFLD, neither at one year (38.1% vs. 48.4%; p = 0.249) nor at five (64.5% vs. 57.0%; p = 0.363) years. However, carriers of the variant had reduced 5-year transplant-free survival, indepen- dently of the etiology (63.4% vs. 80.7%; log-R 5.3; p = 0.021 for ALD/NAFLD patients) (38.2% vs. 74.7%; log-R 9.0; p = 0.003 for viral hepatitis). Alternatively, we analyzed the interactions with the variant rs738409 (PNPLA3) in a subcohort of 294 patients. This polymorphism was in agreement with the Hardy–Weinberg equilibrium (p > 0.05). The result showed that the negative effect of the rs72613567-TA variant was present among carriers of the rs738409-GG genotype, who had a greater risk of liver-related mortality [aHR:8.550 (95% CI 1.490–21.864); p < 0.001], ascites [aHR:11.048 (3.83–32.129); p < 0.001], and HCC [aHR:3.641 (1.019–13.160); p = 0.048]. By contrast, this effect was diluted in non-carriers of the rs738409-GG genotype, and none of the outcomes were found to be significantly changed, although the rs72613567-TA/TA was still not protective. The cumulative incidences of these events are shown in Figure 3A–C. TA variant was present among carriers of the rs738409-GG genotype, who had a greater risk of liver-related mortality [aHR:8.550 (95% CI 1.490–21.864); p < 0.001], ascites [aHR:11.048 (3.83–-32.129); p < 0.001], and HCC [aHR:3.641 (1.019–13.160); p = 0.048]. By contrast, this effect was diluted in non-carriers of the rs738409-GG genotype, and none of the outcomes were found to be significantly changed, although the rs72613567-TA/TA Int. J. Mol. Sci. 2022, 23, 11840 6 of 11 was still not protective. The cumulative incidences of these events are shown in Figure 3A–C. A PNPLA3 GG PNPLA3 allele C 100 100 rs72613567 T/T-T/TA Liver transplantation or rs72613567 T/T-T/TA log-Rank=5.2; p = 0.023 ns rs72613567 TA/TA liver-related death rs72613567 TA/TA aHR = 8.5; p < 0.001 Probability Probability 50 50 0 0 0 12 24 36 48 60 0 12 24 36 48 60 Time (months) Time (months) T/T-T/TA 116 102 88 73 63 51 T/T-T/TA 159 133 1 10 88 73 50 TA/TA 7 5 5 4 3 1 TA/TA 12 10 7 7 6 4 B 100 rs72613567 T/T-T/TA log-Rank = 16.5; p < 0.001 100 rs72613567 T/T-T/TA ns rs72613567 TA/TA aHR = 11.1; p < 0.001 rs72613567 TA/TA Probability Probability Ascites 50 50 0 0 0 12 24 36 48 60 0 12 24 36 48 60 Time (months) Time (months) T/T-T/TA 116 98 81 67 53 37 T/T-T/TA 159 128 101 82 65 43 TA/TA 7 4 2 2 2 1 TA/TA 12 10 6 6 5 3 C 100 rs72613567 T/T-T/TA log-Rank = 4.7; p = 0.031 100 rs72613567 T/T-T/TA aHR = 3.6; p = 0.048 ns rs72613567 TA/TA rs72613567 TA/TA Probability Probability HCC 50 50 0 0 0 12 24 36 48 60 0 12 24 36 48 60 Time (months) Time (months) T/T-T/TA 116 101 89 72 59 44 T/T-T/TA 159 155 132 105 82 46 TA/TA 7 5 4 3 3 1 TA/TA 12 12 10 7 7 4 Figure 3. Effect of the PNPLA3 rs738409 GG genotype on the risk of (A) liver-related mortality, (B) ascites, and (C) hepatocellular carcinoma, according to the HSD17B13 rs72613567 genotype. aHR: adjusted hazard ratio; ns: not significant p-value. 3. Discussion Several studies have revealed an association of epidemiological factors (geographic location, race, and environment), underlying different genetic backgrounds, with the prevalence of chronic liver diseases. Some of these genetic factors correlate with disease severity, progression to HCC, and liver-related outcomes. Usually, these mutations lead to structural, functional, or expression changes in the protein they code for, influencing a metabolic pathway that is differentially activated in disease progression. However, the liver, the most metabolically active and ductile organ, can go through different metabolic stages throughout liver disease progression, for the maintenance of hepatic regeneration and homeostasis [15]. Moreover, the physiopathology usually expands beyond the liver, Int. J. Mol. Sci. 2022, 23, 11840 7 of 11 and the consequences of these variants in peripheric tissues may also account for some of the risks, although they have not been properly evaluated yet. The HSD17B13 gene encodes for 17-beta-hydroxysteroid dehydrogenase 13, a retinol dehydrogenase highly expressed in the liver. Although its function is not completely clear, it is associated with lipid droplets, and it appears to be involved in the metabolism of lipids, as well as of steroid hormones, prostaglandins, xenobiotics, and retinoids [16]. The PNPLA3 protein also localizes to lipid droplets’ surfaces and plays a role in retinol metabolism; the I148M variant leads to increased fat accumulation in hepatic stellate cells, the main cellular site of retinol in the body. Retinol contributes to the regulation of gene transcription by binding nuclear hormone receptors (RAR/RXR), and the impairment induced by the variant in PNPLA3 is associated with a proinflammatory and profibrogenic phenotype [17]. Contrariwise, although the variant in HSD17B13 was linked with decreased levels of the spliced variant, it has been associated with protection against alcoholic and non-alcoholic fatty liver disease and against the progression towards fibrosis and HCC [18]. However, the findings of this work suggest that the variant rs72613567:TA in HSD17B13 has no protective effect, but indeed increases the risk of decompensation and death in patients with advanced chronic liver disease. Previously, Ting et al. discussed the pos- sibility of losing the protective effect given by the mutation once advanced fibrosis de- velops [19]; Scheiner et al. [14] showed, in agreement with our results, that the variant was not protective against hepatic decompensation and mortality among patients with portal hypertension. Numerous studies reported associations between genetics and hepatic decompensations [20,21]; nevertheless, for the first time, as far as we are concerned, we report here that the effect of the variant might change according to the stage of the disease. Of note, most studies have been conducted on patients with ALD/NAFLD. Our cohort included few NAFLD patients (4.3% of total), and 42% were viral hepatitis patients, a prevalence that is consistent with epidemiological studies from the same period as this study [22], in which consecutive patients from outpatient clinics were included. In agreement with other studies, we showed that the underlying etiology of cirrhosis could impact the effect of the mutation on the prognosis, with a higher risk in patients with viral hepatitis. Certain differences in the pathophysiology could possibly explain this, such as the HCV lifecycle being closely linked to liver lipid metabolism [23]. In HBV patients, the mutation in PNPLA3 has been associated with treatment response [24]. Further studies examining the interactions between 17-beta-HSD13 and the steps of HBV and HCV infection are required. In any case, the variant in HSD17B13 did not show significant protection against liver-related outcomes in ALD/NAFLD patients. Interestingly, the negative impact of the HSD17B13 T>TA variant was diluted among non-carriers of the GG genotype of rs738409, suggesting an interaction between both polymorphisms, as previously reported [25,26]. Previous data showed a reduced effect of these variants on circulating retinol levels in patients with advanced chronic liver disease, in comparison to earlier stages [14]. Another study also observed that the impact of the variant in PNPLA3 was different depending on the stage of the disease and the etiology [8]. These findings highlight the role of lipid droplets and retinol metabolites, as well as the fact that the metabolic plasticity of the liver is needed to maintain homeostasis through different stages of liver disease, and lead us to hypothesize that genetic variants disrupting these pathways may have a singular impact in the context of end-stage liver disease. In this context, the recent literature revealed some of the metabolic abnormalities taking place in liver cells of compensated and decompensated cirrhotic patients, such as impaired oxidative phosphorylation and fatty acid hypometabolism due to the inhibition of beta-oxidation [27]. Genetics is now considered a promising approach for the development of precision medicine, with great clinical applications for improving liver disease risk stratification, especially in NAFLD/ALD patients. There is also interest in exploring the pharmaceutical inhibition of 17-beta-HSD13 in order to diminish its steatogenic effect and prevent liver damage, just as the rs72613567:TA variant does. However, future studies analyzing the mechanistic effect of the genotype of HSD17B13 (and other variants of interest) in different Int. J. Mol. Sci. 2022, 23, 11840 8 of 11 stages of the disease are warranted before being considered therapeutic targets with wide clinical applications. This study has some limitations, mainly due to its retrospective nature. Nevertheless, we included a well-characterized, homogeneous cohort of cirrhotic patients that allowed us to assess the effect of the mutations in a specific setting. The majority of studies so far were population-based and limited in adjusting the results for liver function. Therefore, our sample size was modest compared to other studies, although a fair number of cases were included for some specific endpoints. Moreover, we only included individuals from our country; thus, the findings may not necessarily be extrapolated to other ethnicities, although the allele frequency observed here was similar to other studies using broader populations. Nevertheless, we acknowledge a deviation in HWE for HSD17B13; despite the possibility that it could be linked to methodological errors or non-random mating or sampling, this was taken into account, and it could indeed reflect an association with the disease. In conclusion, the results of this study suggest that the protective effect conferred by the carriage of the HSD17B13 rs72613567:TA genotype for liver disease may change in the context of cirrhosis, where it increased the risk of decompensation and death during the follow-up. A deeper understanding of the metabolic changes throughout the stages of advanced liver disease, focusing on the underlying effects of the HSD17B13 genotype, is indispensable. 4. Materials and Methods 4.1. Experimental Design Study population: A total of 483 patients suffering from liver cirrhosis of different etiologies were enrolled from 2007–2016 at the outpatient clinics of four different hospitals: Virgen del Rocio University Hospital, Valme University Hospital (Seville, Spain), Santa Creu i Sant Pau Hospital (Barcelona, Spain), and General University Hospital of Alicante (Alicante, Spain). The exclusion criteria were as follows: age < 18 years; absence of signed informed consent; active alcohol consumption; previous liver transplant or transjugular intrahepatic portosystemic shunt procedure; hepatocellular carcinoma diagnosed within the first 6 months of follow-up; and the presence of comorbidities with an estimated survival of less than 6 months at the time of enrollment. Cirrhosis was diagnosed according to the clinical practice guidelines [28]. The study was performed by following ethical guidelines expressed in the Declara- tion of Helsinki, and in the E18 (a guideline on genomic sampling and the management of genomic data) and E6 (a guideline for good clinical practice) from the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. Demographic, clinical, and epidemiological data were recorded at the first visit, and all data were coded in the original clinical database. Human blood samples were collected after obtaining a signed informed consent form, as approved by the ethical committee of each hospital. Definition of hepatic decompensation and transplant-free survival: Patients’ medical records were reviewed for the following events that defined (further) hepatic decompensa- tion at baseline and during follow-up: large-volume paracentesis, spontaneous bacterial peritonitis (SBP), esophageal variceal bleeding (EVB), overt HE (West Haven grade > 1), hepatorenal syndrome, and liver-related death. Any (further) hepatic decompensation was defined by having at least one of these events occurring during follow-up. HCC was diagnosed without biopsy in the majority of the cases, because current clinical diagnostic approaches, including ultrasonography, computed tomography, or magnetic resonance imaging, were sufficient to diagnose HCC. The patients were followed-up for up to five years, being censored on the day of death, or the day of the surgery if they were transplanted, for the calculation of transplant- free survival. Liver- and non-liver-related death and liver transplants were considered competing events for hepatic decompensation and HCC development. Int. J. Mol. Sci. 2022, 23, 11840 9 of 11 4.2. DNA Genotyping Genomic DNA was extracted from each sample of peripheral blood by using a MagNA Pure Compact Nucleic Acid Isolation Kit I. The concentration and purity of the DNA were evaluated by NanoDrop (ThermoFisher, Waltham, MA, USA). The genotyping of the single- nucleotide polymorphisms (SNP) of interest, PNPLA3 rs738409 (assay ID: C_7241_10) and HSD17B13 rs72613567 (custom TaqMan assay design), was performed using TaqMan SNP genotyping assays and chemistries (ThermoFisher, USA). PCRs were carried out, following the amplification protocol of FastStart Essential DNA Probes Master (Roche, Basel, Switzerland), in a QuantStudio 1 Real-Time PCR (ThermoFisher, USA). Genotype distribution was assessed for Hardy–Weinberg equilibrium by using a goodness-of-fit chi-square test; p > 0.05 was indicative of agreement with the equilibrium. 4.3. Statistical Analysis Categorical variables are presented as percentages, while continuous variables are presented as means ± standard deviation. The survival analysis was assessed by means of Cox regression for continuous variables, and by the Kaplan–Meier method, comparing subgroups by using the Mantel–Cox statistic, for categorical data. Adjusted multivariate analyses for the different outcomes were assessed by Cox regression, including age, sex, and variables with a known impact on prognosis, such as liver function measured by MELD score and etiology-dependent factors. These factors included the achievement of a sustained virological response for viral hepatitis, or the moderate relapse in alcohol intake for alcoholic hepatitis (any severe relapse leading to hospitalization was retrospectively excluded from the analysis). The strength of association between the variables and the outcomes under each model was represented by hazard ratios with 95% CIs. Missing data that were considered as missing completely at random biologically implausible values were set to missing values. Variables with <5% of missing data were imputed by multiple lineal regression, and variables with >5% of missing data were dropped. A two-sided p-value of <0.05 was set as the threshold for statistical significance. SPSS (v.25.0; SPSS, Inc., Chicago, IL, USA) and STATA (v.16, StataCorp LLC, College Station, TX, USA) statistical packages were used for analyses, and graphics were performed by using a GraphPad PRISM (v.8.0, GraphPad Software, Inc., San Diego, CA, USA). Author Contributions: A.G.-G. led the formal analysis and writing—original draft; Á.R. contributed to the data curation and supported the formal analysis; M.R.G.-L. contributed to the data curation and acquisition; R.M.-H. supported the formal analysis and methodology; R.G.-D. equally contributed to the data curation, and led the resources; D.M.-M. contributed to the data curation and methodology; R.M.-V., S.G. and J.G., equally contributed to the resources and methodology; R.F. and G.S. equally contributed to the validation; J.A. supported the conceptualization, led the supervision, and con- tributed to funding acquisition; M.R.-G. led the conceptualization, contributed to funding acquisition, and led the writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: The research leading to these results received funding from Consejería de Salud de la Junta de Andalucía under grant agreements PE-0451-2018 and P20_01075 del programa PAIDI 2020 and from Instituto de Salud Carlos III, Ministerio de Ciencia y Competitividad under grant agreements PI19/01404 and PI19/00589. This research was also funded by European project LITMUS 777377 from Horizon 2020; a contract with fellowships from Consejería de Salud y Familias, Junta de Andalucía, supporting Antonio Gil-Gómez (RH-122-2020) and Angela Rojas (RH-002-2021); grant agreements PFIS FI20/00201 and IFI18/00041 from Instituto de Salud Carlos III to support Sheila Gato and Rocio Montero-Vallejo; and by support from Talento Doctores (PID Junta Andalucía, DOC_00866) to Rocío Muñoz-Hernández. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee “CEI Virgen Macarena-Virgen del Rocío” (protocol C330020). Int. J. Mol. Sci. 2022, 23, 11840 10 of 11 Informed Consent Statement: Written informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to privacy reasons. Conflicts of Interest: The authors declare no conflict of interest. References 1. Cheemerla, S.; Balakrishnan, M. Global Epidemiology of Chronic Liver Disease. Clin. Liver Dis. 2021, 17, 365–370. [CrossRef] [PubMed] 2. Sepanlou, S.G.; Safiri, S.; Bisignano, C.; Ikuta, K.S.; Merat, S.; Saberifiroozi, M.; Poustchi, H.; Tsoi, D.; Colombara, D.V.; Abdoli, A.; et al. The global, regional, and national burden of cirrhosis by cause in 195 countries and territories, 1990–2017: A systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol. Hepatol. 2020, 5, 245–266. [CrossRef] 3. Asrani, S.K.; Devarbhavi, H.; Eaton, J.; Kamath, P.S. Burden of liver diseases in the world. J. Hepatol. 2019, 70, 151–171. [CrossRef] [PubMed] 4. Luukkonen, P.K.; Qadri, S.; Ahlholm, N.; Porthan, K.; Männistö, V.; Sammalkorpi, H.; Penttilä, A.K.; Hakkarainen, A.; Lehtimäki, T.E.; Gaggini, M.; et al. Distinct contributions of metabolic dysfunction and genetic risk factors in the pathogenesis of non-alcoholic fatty liver disease. J. Hepatol. 2022, 76, 526–535. [CrossRef] [PubMed] 5. Loomba, R.; Schork, N.; Chen, C.H.; Bettencourt, R.; Bhatt, A.; Ang, B.; Nguyen, P.; Hernandez, C.; Richards, L.; Salotti, J.; et al. Heritability of Hepatic Fibrosis and Steatosis Based on a Prospective Twin Study. Gastroenterology 2015, 149, 1784–1793. [CrossRef] [PubMed] 6. Buch, S.; Stickel, F.; Trépo, E.; Way, M.; Herrmann, A.; Nischalke, H.D.; Brosch, M.; Rosendahl, J.; Berg, T.; Ridinger, M.; et al. A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis. Nat. Genet. 2015, 47, 1443–1448. [CrossRef] 7. Stickel, F.; Buch, S.; Nischalke, H.D.; Weiss, K.H.; Gotthardt, D.; Fischer, J.; Rosendahl, J.; Marot, A.; Elamly, M.; Casper, M.; et al. Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis. Am. J. Gastroenterol. 2018, 113, 1475–1483. [CrossRef] 8. Mandorfer, M.; Scheiner, B.; Stättermayer, A.F.; Schwabl, P.; Paternostro, R.; Bauer, D.; Schaefer, B.; Zoller, H.; Peck- Radosavljevic, M.; Trauner, M.; et al. Impact of patatin-like phospholipase domain containing 3 rs738409 G/G genotype on hepatic decompensation and mortality in patients with portal hypertension. Aliment. Pharmacol. Ther. 2018, 48, 451–459. [CrossRef] 9. Trépo, E.; Caruso, S.; Yang, J.; Imbeaud, S.; Couchy, G.; Bayard, Q.; Letouzé, E.; Ganne-Carrié, N.; Moreno, C.; Oussalah, A.; et al. Common genetic variation in alcohol-related hepatocellular carcinoma: A case-control genome-wide association study. Lancet Oncol. 2022, 23, 161–171. [CrossRef] 10. Abul-Husn, N.S.; Cheng, X.; Li, A.H.; Xin, Y.; Schurmann, C.; Stevis, P.; Liu, Y.; Kozlitina, J.; Stender, S.; Wood, G.C.; et al. A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease. N. Engl. J. Med. 2018, 378, 1096–1106. [CrossRef] 11. Tang, S.; Zhang, J.; Mei, T.T.; Zhang, W.Y.; Zheng, S.J.; Yu, H.B. Association of HSD17B13 rs72613567: TA allelic variant with liver disease: Review and meta-analysis. BMC Gastroenterol. 2021, 21, 490. [CrossRef] [PubMed] 12. Stickel, F.; Lutz, P.; Buch, S.; Nischalke, H.D.; Silva, I.; Rausch, V.; Fischer, J.; Weiss, K.H.; Gotthardt, D.; Rosendahl, J.; et al. Genetic Variation in HSD17B13 Reduces the Risk of Developing Cirrhosis and Hepatocellular Carcinoma in Alcohol Misusers. Hepatology 2020, 72, 88–102. [CrossRef] [PubMed] 13. Yang, J.; Trépo, E.; Nahon, P.; Cao, Q.; Moreno, C.; Letouzé, E.; Imbeaud, S.; Bayard, Q.; Gustot, T.; Deviere, J.; et al. A 17-Beta- Hydroxysteroid Dehydrogenase 13 Variant Protects From Hepatocellular Carcinoma Development in Alcoholic Liver Disease. Hepatology 2019, 70, 231–240. [CrossRef] 14. Scheiner, B.; Stättermayer, A.F.; Schwabl, P.; Bucsics, T.; Paternostro, R.; Bauer, D.; Simbrunner, B.; Schmidt, R.; Marculescu, R.; Ferlitsch, A.; et al. Impact of HSD17B13 rs72613567 genotype on hepatic decompensation and mortality in patients with portal hypertension. Liver Int. 2020, 40, 393–404. [CrossRef] [PubMed] 15. Michalopoulos, G.K. Hepatostat: Liver regeneration and normal liver tissue maintenance. Hepatology 2017, 65, 1384–1392. [CrossRef] [PubMed] 16. Ma, Y.; Belyaeva, O.V.; Brown, P.M.; Fujita, K.; Valles, K.; Karki, S.; de Boer, Y.S.; Koh, C.; Chen, Y.; Du, X.; et al. 17-Beta Hydroxysteroid Dehydrogenase 13 Is a Hepatic Retinol Dehydrogenase Associated with Histological Features of Nonalcoholic Fatty Liver Disease. Hepatology 2019, 69, 1504–1519. [CrossRef] [PubMed] 17. Mondul, A.; Mancina, R.M.; Merlo, A.; Dongiovanni, P.; Rametta, R.; Montalcini, T.; Valenti, L.; Albanes, D.; Romeo, S. PNPLA3 I148M Variant Influences Circulating Retinol in Adults with Nonalcoholic Fatty Liver Disease or Obesity. J. Nutr. 2015, 145, 1687–1691. [CrossRef] [PubMed] 18. Pirola, C.J.; Garaycoechea, M.; Flichman, D.; Arrese, M.; San Martino, J.; Gazzi, C.; Castaño, G.O.; Sookoian, S. Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease. J. Lipid Res. 2019, 60, 176–185. [CrossRef] Int. J. Mol. Sci. 2022, 23, 11840 11 of 11 19. Ting, Y.W.; Kong, A.S.; Zain, S.M.; Chan, W.K.; Tan, H.L.; Mohamed, Z.; Pung, Y.F.; Mohamed, R. Loss-of-function HSD17B13 variants, non-alcoholic steatohepatitis and adverse liver outcomes: Results from a multi-ethnic Asian cohort. Clin. Mol. Hepatol. 2021, 27, 486–498. [CrossRef] 20. Grimaudo, S.; Pipitone, R.M.; Pennisi, G.; Celsa, C.; Cammà, C.; Di Marco, V.; Barcellona, M.R.; Boemi, R.; Enea, M.; Gian- netti, A.; et al. Association Between PNPLA3 rs738409 C>G Variant and Liver-Related Outcomes in Patients with Nonalcoholic Fatty Liver Disease. Clin. Gastroenterol. Hepatol. 2020, 18, 935–944.e3. [CrossRef] 21. Gil-Gómez, A.; Ampuero, J.; Rojas, Á.; Gallego-Durán, R.; Muñoz-Hernández, R.; Rico, M.C.; Millán, R.; García-Lozano, R.; Francés, R.; Soriano, G.; et al. Development and Validation of a Clinical-Genetic Risk Score to Predict Hepatic Encephalopathy in Patients with Liver Cirrhosis. Am. J. Gastroenterol. 2021, 116, 1238–1247. [CrossRef] [PubMed] 22. Pimpin, L.; Cortez-Pinto, H.; Negro, F.; Corbould, E.; Lazarus, J.V.; Webber, L.; Sheron, N. Burden of liver disease in Europe: Epidemiology and analysis of risk factors to identify prevention policies. J. Hepatol. 2018, 69, 718–735. [CrossRef] [PubMed] 23. Hofmann, S.; Krajewski, M.; Scherer, C.; Scholz, V.; Mordhorst, V.; Truschow, P.; Schöbel, A.; Reimer, R.; Schwudke, D.; Herker, E. Complex lipid metabolic remodeling is required for efficient hepatitis C virus replication. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2018, 1863, 1041–1056. [CrossRef] [PubMed] 24. Enomoto, H.; Aizawa, N.; Hasegawa, K.; Ikeda, N.; Sakai, Y.; Yoh, K.; Takata, R.; Yuri, Y.; Kishino, K.; Shimono, Y.; et al. Possible Relevance of PNPLA3 and TLL1 Gene Polymorphisms to the Efficacy of PEG-IFN Therapy for HBV-Infected Patients. Int. J. Mol. Sci. 2020, 21, 3089. [CrossRef] [PubMed] 25. Gellert-Kristensen, H.; Richardson, T.G.; Davey Smith, G.; Nordestgaard, B.G.; Tybjaerg-Hansen, A.; Stender, S. Combined Effect of PNPLA3, TM6SF2, and HSD17B13 Variants on Risk of Cirrhosis and Hepatocellular Carcinoma in the General Population. Hepatology 2020, 72, 845–856. [CrossRef] [PubMed] 26. Seko, Y.; Yamaguchi, K.; Mizuno, N.; Okuda, K.; Takemura, M.; Taketani, H.; Hara, T.; Umemura, A.; Nishikawa, T.; Moriguchi, M.; et al. Combination of PNPLA3 and TLL1 polymorphism can predict advanced fibrosis in Japanese patients with nonalcoholic fatty liver disease. J. Gastroenterol. 2018, 53, 438–448. [CrossRef] [PubMed] 27. Arroyo, V.; Moreau, R.; Jalan, R. Acute-on-Chronic Liver Failure. N. Engl. J. Med. 2020, 382, 2137–2145. [CrossRef] 28. European Association for the Study of the Liver; Clinical Practice Guideline Panel. EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis—2021 update. J. Hepatol. 2021, 75, 659–689. [CrossRef]