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Clinical Infection & Immunity

Original Article

Volume 8, Number 1, March 2023, pages 24-30

PNPLA3 and HSD17B13 Polymorphisms’ Influence on Liver Fibrosis Development in a Small Cohort of Italian Patients With Viral Hepatitis

The hepatitis C virus (HCV) and the hepatitis B virus (HBV), in single or dual infection, affect millions of people worldwide, and despite antiviral treatments which have clearly improved the history of chronic viral liver disease can still lead to severe fibrosis and cirrhosis [1, 2]. Liver steatosis is another histological lesion that can be present in viral hepatitis, especially due to HCV [3].

The pathogenesis of liver damage can be influenced by the genetic background of patients. Although single nucleotide polymorphisms (SNPs) of many genes have been implicated in the development of liver steatosis and/or fibrosis, the specific involvement of each polymorphism remains often unclear. Among these genetic factors, HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, and the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, involved in lipolytic and lipogenic activity in the liver and adipose tissue, feature prominently.

The PNPLA3 variant (rs738409 C>G) has been evaluated in many studies and has been associated with hepatic steatosis and fibrosis in nonalcoholic fatty liver disease (NAFLD) and viral hepatitis [4-12]. However, in a recent study on a Japanese cohort, mutated rs738409 genotype showed an insignificant effect in patients with viral cirrhosis, while it was significantly associated to cirrhosis in patients with NAFLD and alcoholic liver disease [13].

More recently, the HSD17B13 gene rs72613567 TA allelic variant has been associated with a reduced risk of developing liver fibrosis and cirrhosis in patients with NAFLD, viral hepatitis and alcoholic liver disease [14-16, 17]; controversial data are emerging about the effect of HSD17B13 gene variant on hepatocellular carcinoma (HCC) development, seeming protective in alcoholic liver disease [18], and favoring in HCV-related cirrhosis [19]. Moreover, polymorphisms of different genes can positively or negatively influence their expression. For example, steatogenic alleles of PNPLA3 and transmembrane 6 superfamily member 2 (TM6SF2) gene, related to high risk of fatty liver disease, amplify the alanine aminotransferases (ALT)-lowering effect of the HSD17B13 variant [15], while the SERPINA 1Pi*Z variant concurs in reducing the risk of liver cirrhosis by HSD17B13 [16]. Different studies have involved populations in Northern and Eastern Europe and the USA [14-16], but recently Kubiliun et al focalized on the role of gene polymorphisms in populations of different ethnicity, explaining, at least partially, differences in risk of NAFLD [20].

In this study we evaluated the possible correlation of PNPLA3 and HSD13B17 polymorphisms with liver cirrhosis in patients with single or dual HBV and HCV infections from Italy.

Two hundred eighty patients with chronic liver disease were enrolled in the study: 73.6% had chronic infection with HCV (anti-HCV/HCV RNA-positive), 16% with HBV (hepatitis B surface antigen (HBsAg)/HBV DNA-positive) and 8.6% with both HBV and HCV (HBsAg/anti-HCV/HCV RNA-positive) (Table 1). In the co-infected group, 30 patients showed both HBV and HCV detectable viral load, seven only HBV and five only HCV viral load at time of observation.

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Table 1. General Characteristics of the Study Population

Patients had been followed up at one of three Liver Units in the Campania region (southern Italy) which have cooperated for years [21, 22] and participated at the present study.

Most patients were asymptomatic and had never experienced episodes of liver decompensation. Patients were naive from antiviral treatment at the time of our evaluation; none of the patients had history of ongoing intravenous drug or alcohol abuse (< 30 g/day for female and 40 g/day for males) in the previous 6 months.

The stage of liver disease was assessed by percutaneous liver biopsy, according to Ishak et al [23] or by transient elastography (TE, Fibro Scan^®, EchoSens, Paris, France). In particular, 173 patients underwent liver biopsy and 107 transient elastography. We defined as patients with liver cirrhosis those with a histological fibrosis score of 6 or with ≥ 12 kPa at elastography.

Liver function tests and HSD17B13/PNPLA3 polymorphisms were analyzed in all patients. Samples of serum and whole blood were obtained for each patient at the time of liver fibrosis evaluation and stored at -80 °C.

The study was approved by the Ethics Committee of the Azienda Ospedaliera University of the Second University of Naples (number: 214/2012), now University of Campania “L. Vanvitelli”. Patients gave their informed consent to the anonymous use of their clinical data and collection and storage of blood samples. The study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki Declaration.

HCV, HBV, hepatitis D virus (HDV) and human immunodeficiency virus (HIV) serum markers and liver function tests were performed with routine methods in use at our hospital.

HCV genotype was determined using HCV genotype assay (Lipa) (Bayer, France). Circulating HCV RNA and HBV DNA were quantified by a real-time polymerase chain reaction (PCR) in a LightCycler 1.5 (Roche Diagnostics, Branchburg, NJ, USA) as previously described [22].

Genomic DNA was extracted from peripheral whole blood with a DNA extraction kit (Promega, Madison WI, USA). All individuals were genotyped for the SNP rs72613567:TA allele to identify an adenine insertion causing a splice variant, using a TaqMan allelic discrimination custom assay (ID: ANNKVTJ) (Applied Biosystems, USA) on ABI 7900HT Real Time PCR system. Moreover, the same patients were also genotyped for PNPLA3 (I148M) rs738409 using pre-designed assay primers and probes purchased from Applied Biosystems (Foster City, CA, USA).

Continuous variables were summarized as median and range, while categorical variables as absolute and relative frequencies. Differences in the categorical variables were evaluated by Pearson chi-square test. Univariate analysis was performed by Fisher’s exact test, and a general linear model was used for multivariable analysis. A P value < 0.05 was considered to be statistically significant. Statistical analysis was performed using SPSS version 20.

Patients were divided into two groups based on the presence (112 patients) or absence (168 patients) of liver cirrhosis. All patients were Caucasian and HDV and HIV-negative.

Two hundred ten (75%) patients had increased ALT serum levels and 70 (25%) had normal ALT.

The distribution of HBV and HCV viremia, HCV genotypes and HSD17B13 and PNPLA3 polymorphisms are shown in Table 1.

PNPLA3 mutants (hetero- and homozygous) were prevalent in our population, while most of the patients were wild type (WT) for the HSD17B13 gene (Table 1).

The PNPLA3 mutant correlated with elevated aspartate aminotransferase (AST) and ALT levels (P = 0.03 and 0.005, respectively), and the HSD17B13 mutant with elevated HCV RNA serum levels (P = 0.04). HBV DNA levels and HCV genotypes did not show any correlation with the polymorphisms studied (HBV DNA vs. PNPLA3 (WT; heterozygote type (HT) + mutant type (MT)) P = 0.54; HBV DNA vs. HSD17B13 (WT; HT + MT) P = 0.54; HCV genotypes (genotype 1; other) PNPLA3 (WT; HT + MT) P = 0.68; HCV genotype (gen1; other) HSD17B13 (WT; HT + MT) P = 0.91). Fibrosis stage was not influenced by viral etiology.

PNPLA3 mutants were significantly associated with the absence of cirrhosis in the global study population (P = 0.01) and in the HCV-infected group (P < 0.001), while HSD17B13 mutants were associated with the absence of cirrhosis in the HCV-infected patients only (P < 0.05; Table 2).

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Table 2. Effect of PNPLA3 or HSD17B13 on Liver Cirrhosis in All Patients and in HCV-Infected Patients

At univariable analysis, age was a factor favoring cirrhosis while PNPLA3 mutants were negatively associated with liver cirrhosis (P < 0.01 and 0.017, respectively; Table 3). At multivariable analysis, only age was confirmed as an independent factor promoting liver cirrhosis (odds ratio (OR): 7.79; P ≤ 0.001) (Table 3).

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Table 3. Analysis of the Variables That Impact Cirrhosis Outcome in the Patient Population

To deepen our analysis, we evaluated the possible interference of one polymorphism with the other in the development of liver cirrhosis and found that the presence of any mutant allele, of either PNPLA3 and/or HSD17B13, was weakly negatively associated with liver cirrhosis (P = 0.02) (Table 2); this result was not confirmed at multivariable analysis.

Cirrhosis is the end stage of liver disease; and it is important to identify factors that can positively or negatively influence the progression of hepatic damage. Genetic polymorphisms, such as HSD17B13 and PNPLA3, involved in lipid metabolism and liver steatosis, showed an influence on the natural history of chronic hepatitis and HCC of different etiologies [5-21, 24, 25].

In this study, we analyzed the role of HSD17B13 and PNPLA3 polymorphisms in the development of cirrhosis in patients from Italy with chronic hepatitis due to HBV, HCV, and HBV-HCV.

We did not evaluate liver steatosis because this parameter was not available for patients for whom fibrosis was detected by transient elastography.

In contrast with most previous data, PNPLA3 was not positively associated with liver fibrosis development in all subgroups, while HSD17B13 showed this pattern only in HCV-positive patients. In addition, the presence of any allelic mutants for both gene polymorphisms was also weakly negatively associated with liver fibrosis progression. In contrast with recent data [14-16], these results were not confirmed at multivariable analysis; also, sub-analysis based on different viral etiologies did not give significant results, likely because of the low number of patients, which is the most important limitation of this study. PNPLA3 and HSD17B13 did not influence each other in this population.

Differences related to race and/or nationality of patients might explain the diverse behaviors of HSD17B13 and PNPLA3 polymorphisms. A recent study from Pakistan showed that PNPLA3 was not a major determinant of liver fibrosis in patients with chronic hepatitis C [26]. Similarly, in our previous studies on Italian patients with chronic viral hepatitis of different etiologies, PNPLA3 was associated with liver steatosis, but not with significant fibrosis [7, 8, 21]. Furthermore, in many studies PNPLA3 polymorphism is correlated to steatosis, NAFLD and nonalcoholic steatohepatitis (NASH), which is a trigger to the development of fibrosis [27-29]; unfortunately, we could not evaluate this variable, as data on liver steatosis were available only for half of study patients.

In addition, studies on the role of HSD17B13 in HCC showed that HSD17B13 increased expression is protective for HCC in HBV-positive Asian patients [25], in contrast with what was shown in European patients [14, 18, 24].

Because mechanisms of action of these genes, particularly HSD17B13, have not been completely elucidated, their effects may vary in different contexts [30]; experimental studies have confirmed a possible role of HSD17B13 in the development of liver steatosis either in over-expression or in under-expression of the gene [31, 32], while experiments on PNPLA3 expression on activation of hepatic stellate cells (HSCs) have suggested that PNPLA3 induction does not appear to be required for the fibrogenic phenotype of HSCs and its downregulation resulted in increased expression of profibrogenic factors regardless of the PNPLA3 genotype [33].

In conclusion, in our Italian population PNPLA3 and HSD17B13 polymorphisms were not positively associated with fibrosis progression in chronic viral hepatitis.

Acknowledgments

The authors thank Ms. Patrizia Cirillo and Mr. Giovanni Di Napoli for their nursing assistance.

Financial Disclosure

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of Interest

All the authors of the manuscript declare that they have no conflicts of interest relevant to this paper.

Informed Consent

Informed consent was obtained.

Author Contributions

Study conception and design: RZ, NC. Experimental procedures: GC, DI, MS. AM, MV, SDP, MM, and MI obtained clinical data and built the database. RZ, NC, LEA, and ED-M analyzed data. RZ, NC, LEA, ED-M, and EMdG interpreted data. RZ and NC wrote the manuscript. All authors approved the final manuscript.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

1. Polaris Observatory HCVC. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol Hepatol. 2017;2(3):161-176.
   doi
2. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386(10003):1546-1555.
   doi
3. Adinolfi LE, Gambardella M, Andreana A, Tripodi MF, Utili R, Ruggiero G. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specific HCV genotype and visceral obesity. Hepatology. 2001;33(6):1358-1364.
   doi pubmed
4. Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet. 2008;40(12):1461-1465.
   doi pubmed
5. Valenti L, Nobili V, Al-Serri A, Rametta R, Leathart JB, Zappa MA, Dongiovanni P, et al. The APOC3 T-455C and C-482T promoter region polymorphisms are not associated with the severity of liver damage independently of PNPLA3 I148M genotype in patients with nonalcoholic fatty liver. J Hepatol. 2011;55(6):1409-1414.
   doi pubmed
6. Valenti L, Rumi M, Galmozzi E, Aghemo A, Del Menico B, De Nicola S, Dongiovanni P, et al. Patatin-like phospholipase domain-containing 3 I148M polymorphism, steatosis, and liver damage in chronic hepatitis C. Hepatology. 2011;53(3):791-799.
   doi pubmed
7. Zampino R, Coppola N, Cirillo G, Boemio A, Grandone A, Stanzione M, Capoluongo N, et al. Patatin-like phospholipase domain-containing 3 I148M variant is associated with liver steatosis and fat distribution in chronic hepatitis B. Dig Dis Sci. 2015;60(10):3005-3010.
   doi pubmed
8. Zampino R, Coppola N, Cirillo G, Boemio A, Minichini C, Marrone A, Stanzione M, et al. Insulin resistance and steatosis in HBV-HCV co-infected patients: Role of PNPLA3 polymorphisms and impact on liver fibrosis progression. World J Hepatol. 2014;6(9):677-684.
   doi pubmed
9. Serper M, Vujkovic M, Kaplan DE, Carr RM, Lee KM, Shao Q, Miller DR, et al. Validating a non-invasive, ALT-based non-alcoholic fatty liver phenotype in the million veteran program. PLoS One. 2020;15(8):e0237430.
   doi pubmed
10. Mazo DF, Malta FM, Stefano JT, Salles APM, Gomes-Gouvea MS, Nastri ACS, Almeida JR, et al. Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population. Ann Hepatol. 2019;18(3):466-471.
    doi pubmed
11. Sahlman P, Nissinen M, Puukka P, Jula A, Salomaa V, Mannisto S, Lundqvist A, et al. Genetic and lifestyle risk factors for advanced liver disease among men and women. J Gastroenterol Hepatol. 2020;35(2):291-298.
    doi pubmed
12. De Nicola S, Dongiovanni P, Aghemo A, Cheroni C, D'Ambrosio R, Pedrazzini M, Marabita F, et al. Interaction between PNPLA3 I148M variant and age at infection in determining fibrosis progression in chronic hepatitis C. PLoS One. 2014;9(8):e106022.
    doi pubmed
13. Shao X, Uojima H, Arai T, Ogawa Y, Setsu T, Atsukawa M, Furuichi Y, et al. The risk of cirrhosis and its complications based on PNPLA3 rs738409 G allele frequency. Dig Dis. 2022;40(5):625-634.
    doi pubmed
14. Abul-Husn NS, Cheng X, Li AH, Xin Y, Schurmann C, Stevis P, Liu Y, et al. A protein-truncating HSD17B13 variant and protection from chronic liver disease. N Engl J Med. 2018;378(12):1096-1106.
    doi pubmed
15. Gellert-Kristensen H, Nordestgaard BG, Tybjaerg-Hansen A, Stender S. High risk of fatty liver disease amplifies the alanine transaminase-lowering effect of a HSD17B13 variant. Hepatology. 2020;71(1):56-66.
    doi pubmed
16. Basyte-Bacevice V, Skieceviciene J, Valantiene I, Sumskiene J, Petrenkiene V, Kondrackiene J, Petrauskas D, et al. SERPINA1 and HSD17B13 gene variants in patients with liver fibrosis and cirrhosis. J Gastrointestin Liver Dis. 2019;28(3):297-302.
    doi pubmed
17. Tang S, Zhang J, Mei TT, Zhang WY, Zheng SJ, Yu HB. Association of HSD17B13 rs72613567: TA allelic variant with liver disease: review and meta-analysis. BMC Gastroenterol. 2021;21(1):490.
    doi pubmed
18. Yang J, Trepo E, Nahon P, Cao Q, Moreno C, Letouze E, Imbeaud S, et al. A 17-beta-hydroxysteroid dehydrogenase 13 variant protects from hepatocellular carcinoma development in alcoholic liver disease. Hepatology. 2019;70(1):231-240.
    doi pubmed
19. Burlone ME, Bellan M, Barbaglia MN, Mocchetti G, Mallela VR, Minisini R, Rigamonti C, et al. HSD17B13 and other liver fat-modulating genes predict development of hepatocellular carcinoma among HCV-positive cirrhotics with and without viral clearance after DAA treatment. Clin J Gastroenterol. 2022;15(2):301-309.
    doi pubmed
20. Kubiliun MJ, Cohen JC, Hobbs HH, Kozlitina J. Contribution of a genetic risk score to ethnic differences in fatty liver disease. Liver Int. 2022;42(10):2227-2236.
    doi pubmed
21. Zampino R, Coppola N, Cirillo G, Boemio A, Pisaturo M, Marrone A, Macera M, et al. Abdominal fat interacts with PNPLA3 I148M, but not with the APOC3 variant in the pathogenesis of liver steatosis in chronic hepatitis C. J Viral Hepat. 2013;20(8):517-523.
    doi pubmed
22. Coppola N, Zampino R, Sagnelli C, Bellini G, Marrone A, Stanzione M, Capoluongo N, et al. Cannabinoid receptor 2-63 QQ variant is associated with persistently normal aminotransferase serum levels in chronic hepatitis C. PLoS One. 2014;9(6):e99450.
    doi pubmed
23. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, et al. Histological grading and staging of chronic hepatitis. J Hepatol. 1995;22(6):696-699.
    doi
24. Stickel F, Lutz P, Buch S, Nischalke HD, Silva I, Rausch V, Fischer J, et al. Genetic variation in HSD17B13 reduces the risk of developing cirrhosis and hepatocellular carcinoma in alcohol misusers. Hepatology. 2020;72(1):88-102.
    doi pubmed
25. Chen J, Zhuo JY, Yang F, Liu ZK, Zhou L, Xie HY, Xu X, et al. 17-beta-hydroxysteroid dehydrogenase 13 inhibits the progression and recurrence of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 2018;17(3):220-226.
    doi pubmed
26. Rauff B, Alzahrani B, Chudhary SA, Nasir B, Mahmood S, Bhinder MA, Faheem M, et al. PNPLA3 and TM6SF2 genetic variants and hepatic fibrosis and cirrhosis in Pakistani chronic hepatitis C patients: a genetic association study. BMC Gastroenterol. 2022;22(1):401.
    doi pubmed
27. Unalp-Arida A, Ruhl CE. Patatin-like phospholipase domain-containing protein 3 I148M and liver fat and fibrosis scores predict liver disease mortality in the U.S. population. Hepatology. 2020;71(3):820-834.
    doi pubmed
28. Li G, Tang LJ, Zhu PW, Huang OY, Rios RS, Zheng KI, Chen SD, et al. PNPLA3 rs738409 C>G variant influences the association between visceral fat and significant fibrosis in biopsy-proven nonalcoholic fatty liver disease. J Clin Transl Hepatol. 2022;10(3):439-448.
    doi pubmed
29. Gabriel-Medina P, Ferrer-Costa R, Rodriguez-Frias F, Ciudin A, Augustin S, Rivera-Esteban J, Pericas JM, et al. Influence of type 2 diabetes in the Association of PNPLA3 rs738409 and TM6SF2 rs58542926 Polymorphisms in NASH Advanced Liver Fibrosis. Biomedicines. 2022;10(5):1015.
    doi pubmed
30. Del Vecchio C, Glielmo L, Corless M. Equilibrium and stability analysis of X-chromosome linked recessive diseases model. Proc IEEE Conference on Decision and Control. 2012;6426443:4936-4941.
    doi
31. Adam M, Heikela H, Sobolewski C, Portius D, Maki-Jouppila J, Mehmood A, Adhikari P, et al. Hydroxysteroid (17beta) dehydrogenase 13 deficiency triggers hepatic steatosis and inflammation in mice. FASEB J. 2018;32(6):3434-3447.
    doi pubmed
32. Su W, Wang Y, Jia X, Wu W, Li L, Tian X, Li S, et al. Comparative proteomic study reveals 17beta-HSD13 as a pathogenic protein in nonalcoholic fatty liver disease. Proc Natl Acad Sci U S A. 2014;111(31):11437-11442.
    doi pubmed
33. Rady B, Nishio T, Dhar D, Liu X, Erion M, Kisseleva T, Brenner DA, et al. PNPLA3 downregulation exacerbates the fibrotic response in human hepatic stellate cells. PLoS One. 2021;16(12):e0260721.
    doi pubmed

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