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Reply to: “Lack of hepatic autophagy promotes severity of liver injury but not steatosis”

ATG7 genetic variants behave as fatty liver disease progression modifiers
  • Guido Baselli
    Affiliations
    Precision Medicine – Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; Milan, Italy

    Department of Pathophysiology and Transplantation, Università degli Studi di Milano; Milan, Italy

    SciLifeLab, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden
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  • Author Footnotes
    † Shared senior authors
    Stefano Romeo
    Footnotes
    † Shared senior authors
    Affiliations
    Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, Wallenberg Laboratory, University of Gothenburg; Gothenburg, Sweden

    Clinical Nutrition Unit, Department of Medical and Surgical Science, University Magna Graecia; Catanzaro, Italy

    Department of Cardiology, Sahlgrenska University Hospital; Gothenburg, Sweden
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  • Author Footnotes
    † Shared senior authors
    Luca Valenti
    Correspondence
    Corresponding author. Address: Precision Medicine – Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, via Francesco Sforza 35, 20122, Milan, Italy.
    Footnotes
    † Shared senior authors
    Affiliations
    Precision Medicine – Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico; Milan, Italy

    Department of Pathophysiology and Transplantation, Università degli Studi di Milano; Milan, Italy
    Search for articles by this author
  • Author Footnotes
    † Shared senior authors
Published:August 16, 2022DOI:https://doi.org/10.1016/j.jhep.2022.07.026

      Linked Article

      • Rare ATG7 genetic variants predispose patients to severe fatty liver disease
        Journal of HepatologyVol. 77Issue 3
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          Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver disorders and has a strong heritable component. The aim of this study was to identify new loci that contribute to severe NAFLD by examining rare variants.
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      • Lack of hepatic autophagy promotes severity of liver injury but not steatosis
        Journal of HepatologyVol. 77Issue 5
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          We read with great interest the recently published paper by Baselli et al. in which they identified novel rare autophagy-related 7 (ATG7) genetic variants that were associated with the progression of non-alcoholic fatty liver disease (NAFLD) in the European population.1 The implication of autophagy in regulating lipid metabolism, lipid droplet (LD) biogenesis and NAFLD has been well documented in experimental settings.2–4 However, the direct genetic evidence on autophagy-related genes in human diseases such as NAFLD has been lacking for a long time.
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      To the Editor:
      We read with great pleasure the comment by Ding et al. on our recent study reporting that rare genetic variants impairing the function of autophagy related-7 (ATG7) predispose individuals at risk of fatty liver disease (FLD) associated with metabolic dysfunction (MAFLD) to the development of severe fibrosis and hepatocellular carcinoma.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      ,
      • Ding W.X.
      • Ni H.M.
      • Waguri S.
      • Komatsu M.
      Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
      In our study, we firstly highlighted an enrichment in rare mutations in ATG7 in patients with severe MAFLD compared to healthy individuals. We then validated the impact of rare ATG7 variants on liver disease in the population-based UK Biobank cohort, in a cohort of individuals with metabolic dysfunction, and in a large liver biopsy cohort (LBC). Overall, the low-frequency p.V471A ATG7 and ATG7 mutational burden were associated with hepatocellular ballooning, severe FLD, and hepatocellular carcinoma at a population level.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      We previously showed that hepatic fat accumulation has a causal role in the progression of liver damage,
      • Bianco C.
      • Jamialahmadi O.
      • Pelusi S.
      • Baselli G.
      • Dongiovanni P.
      • Zanoni I.
      • et al.
      Non-invasive stratification of hepatocellular carcinoma risk in non-alcoholic fatty liver using polygenic risk scores.
      and autophagy is involved in hepatic lipid catabolism in hepatocytes.
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      Therefore, an important question was whether the impact of ATG7 variants was mediated by hepatocellular fat accumulation.
      In response to our study, Ding et al. reported that liver-specific Atg5 or Atg7 knockout models were more prone to liver inflammation and fibrosis but were protected from fasting- or partial hepatectomy-induced steatosis,
      • Ding W.X.
      • Ni H.M.
      • Waguri S.
      • Komatsu M.
      Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
      ,
      • Takamura A.
      • Komatsu M.
      • Hara T.
      • Sakamoto A.
      • Kishi C.
      • Waguri S.
      • et al.
      Autophagy-deficient mice develop multiple liver tumors.
      ,
      • Ni H.M.
      • Woolbright B.L.
      • Williams J.
      • Copple B.
      • Cui W.
      • Luyendyk J.P.
      • et al.
      Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.
      suggesting the predisposition to liver damage in carriers of ATG7 mutations was not mediated by intracellular fat accumulation. Consistently, we did not observe any association between p.V471A and other ATG7 variants with steatosis grade in the LBC.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      Furthermore, the p.V471A variant was not associated with steatosis grade in patients at higher risk for FLD progression (Table 1) due to either type 2 diabetes (T2M) or obesity (BMI >30), or otherwise genetically predisposed to FLD (high polygenic risk score for hepatic fat content
      • Bianco C.
      • Jamialahmadi O.
      • Pelusi S.
      • Baselli G.
      • Dongiovanni P.
      • Zanoni I.
      • et al.
      Non-invasive stratification of hepatocellular carcinoma risk in non-alcoholic fatty liver using polygenic risk scores.
      [PRS-HFC]). Conversely, the p.V471A variant was associated with an increased risk of ballooning independently of the steatosis grade.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      These associations were largely confirmed in the UK Biobank, where the p.V471A variant was not associated with steatosis but rather with increased aspartate aminotransferase levels and the loss-of-function variant predisposed severely obese individuals to liver cancer.
      • Ding W.X.
      • Ni H.M.
      • Waguri S.
      • Komatsu M.
      Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
      Table 1Associations between ATG7 p.V471A genotype and liver steatosis grade in subgroup at risk for FLD from the liver biopsy cohort.
      ATG7 genotype
      GroupSample sizeOR (95% CI)adj p value
      T2DM, yes5060.89 (0.49–1.59)0.70
      BMI > 301,2191.09 (0.72–1.65)0.68
      PRS-HFC, High1,0711.18 (0.79–1.75)0.42
      Statistical analysis was performed by ordinal logistic regression using the R software v4.0.3. Models were adjusted by sex and age. When appropriate models were also adjusted by BMI, T2DM, and the PNPLA3 p.I148M, TM6SF2 p.E167K, MBOAT7 rs641738, and GCKR, p.P446L genotypes. All genotypes were investigated under an additive model, p <0.05 were considered statistically significant. ATG7, autophagy-related 7; FLD, fatty liver disease; OR, odds ratio; PRS-HFC, polygenic risk score hepatic fat content; T2M, type 2 diabetes mellitus.
      On the other hand, we confirmed that ATG7 regulates intracellular lipid content in several in vitro models. Indeed, ATG7 KO by RNAi in human hepatoma cell lines and primary hepatocytes facilitated intracellular fat accumulation. Vice versa, overexpression of wild-type ATG7 but not the p.V471A mutant protein protected against lipid accumulation. Similarly, human HepG2 hepatoma cells with stable ATG7 KO or carrying the p.V471A variant in homozygosis showed defective autophagy and were more prone to developing steatosis.
      • Ding W.X.
      • Ni H.M.
      • Waguri S.
      • Komatsu M.
      Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
      Taken together, our data suggest that autophagy protects against steatosis in vitro and are in line with previously published evidence describing lipo-autophagy in hepatocytes.
      • Singh R.
      • Kaushik S.
      • Wang Y.
      • Xiang Y.
      • Novak I.
      • Komatsu M.
      • et al.
      Autophagy regulates lipid metabolism.
      The underlying explanation for the discrepancy between the epidemiological and in vitro findings remains unclear. Of note, ATG7 is strongly conserved in humans and the p.V471A represents the only coding variant with an allelic frequency >0.01 in population studies.
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      In our models, the p.V471A variant resulted in lower ATG7 expression and activity.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      In the LBC, only a handful of patients carried the variant in homozygosis, all of whom displayed liver steatosis. Furthermore, in the Liver-Bible-2021 cohort of individuals with metabolic dysfunction, ATG7 genetic variability was associated with steatosis (controlled attenuation parameter >275db/m, odds ratio 1.90, 95% CI 1.06-3.42, adjusted p = 0.029).
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      Taken together, rare ATG7 variants and the low-frequency p.V471A variant appear to act mostly as modifiers of FLD progression, whereas their impact on hepatic fat accumulation remains elusive. We cannot rule out that ATG7 variants exert a mild predisposition towards steatosis development, as observed in patients with metabolic dysfunction. However, available data suggest that their impact on liver disease progression is not fully accounted for by defective lipo-autophagy leading to the accumulation of intracellular fat. Instead, we observed that defective autophagy leads to the accumulation of p62 and ballooning degeneration.
      • Baselli G.A.
      • Jamialahmadi O.
      • Pelusi S.
      • Ciociola E.
      • Malvestiti F.
      • Saracino M.
      • et al.
      Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
      Indeed, autophagy plays a multifaceted role in hepatocytes, being implicated in lipid droplet turnover but also in the clearance of protein aggregates and damaged mitochondria.
      • Ding W.X.
      • Ni H.M.
      • Waguri S.
      • Komatsu M.
      Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
      Concerning non-parenchymal cells, Atg7 deficiency in hepatic stellate cells protected mice from liver fibrosis,
      • Hernandez-Gea V.
      • Ghiassi-Nejad Z.
      • Rozenfeld R.
      • Gordon R.
      • Fiel M.I.
      • Yue Z.
      • et al.
      Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.
      while Atg7 KO was associated with more severe disease in liver endothelial cells and Kupffer cells.
      • Fukada H.
      • Yamashina S.
      • Izumi K.
      • Komatsu M.
      • Tanaka K.
      • Ikejima K.
      • et al.
      Suppression of autophagy sensitizes Kupffer cells to endotoxin.
      Additional studies are required to clarify: a) the extent to which the impact of the p.V471A variant on liver disease is the result of functional impairment of ATG7 in hepatocytes vs. other cell types; b) to pinpoint the exact mechanism, possibly involving defective clearance of damaged mitochondria and protein aggregates
      • Takamura A.
      • Komatsu M.
      • Hara T.
      • Sakamoto A.
      • Kishi C.
      • Waguri S.
      • et al.
      Autophagy-deficient mice develop multiple liver tumors.
      ,
      • Ni H.M.
      • Woolbright B.L.
      • Williams J.
      • Copple B.
      • Cui W.
      • Luyendyk J.P.
      • et al.
      Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.
      ; c) to clarify the common pathogenic mechanism and differences leading to hepatic and neurological diseases in carriers of ATG7 mutations.

      Financial support

      Italian Ministry of Health (Ministero della Salute), Ricerca Finalizzata RF-2016-02364358 (“Impact of whole exome sequencing on the clinical management of patients with advanced nonalcoholic fatty liver and cryptogenic liver disease”), (LV). Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Ricerca corrente (LV). Fondazione IRCCS Ca’ Granda core COVID-19 Biobank (RC100017A), “Liver BIBLE” (PR-0391) (LV). Innovative Medicines Initiative 2 joint undertaking of European Union’s Horizon 2020 research and innovation programme and EFPIA European Union (EU) Programme Horizon 2020 (under grant agreement No. 777377) for the project LITMUS (LV). The European Union, programme “Photonics” under grant agreement “101016726” (LV). Gilead_IN-IT-989-5790 (LV). Swedish Research Council [Vetenskapsrådet, 2016-01527], SR. Swedish state under the agreement between the Swedish government and the county councils (the ALF-agreement) [SU 2018-04276] (SR). Novo Nordisk Foundation Grant for Excellence in Endocrinology [Excellence Project, 9321-430], the Swedish Diabetes Foundation [DIA 2017-205] (SR). Swedish Heart Lung Foundation [20120533] (SR). Wallenberg Academy Fellows from the Knut and Alice Wallenberg Foundation [KAW 2017.0203] (SR). Astra Zeneca Agreement for Research, Grant SSF ITM17-0384 (SR). Swedish Foundation for Strategic Research, Novo Nordisk Project Grants in Endocrinology & Metabolism - Nordic Region 2020 (SR). AIRC postdoctoral fellowship for abroad [2021- 26794] (GB).

      Authors’ contributions

      LV, SR and GB conceptualized the study. GB performed the analyses. GB and LV drafted the manuscript. LV and SR reviewed the manuscript and supervised the study.

      Conflict of interest

      The authors declare that they have no conflict of interest relevant to the present study. LV has received speaking fees from MSD, Gilead, AlfaSigma and AbbVie, served as a consultant for Gilead, Pfizer, AstraZeneca, Novo Nordisk, Intercept, Diatech Pharmacogenetics and Ionis Pharmaceuticals, and received research grants from Gilead. SR has served as a consultant for AstraZeneca, Celgene, Sanofi, Amgen, Akcea Therapeutics, Camp4, AMbys, Medacorp and Pfizer in the past 5 years, and received research grants from AstraZeneca, Sanofi and Amgen.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following are the supplementary data to this article:

      References

        • Baselli G.A.
        • Jamialahmadi O.
        • Pelusi S.
        • Ciociola E.
        • Malvestiti F.
        • Saracino M.
        • et al.
        Rare ATG7 genetic variants predispose patients to severe fatty liver disease.
        J Hepatol. 2022; 77: 596-606
        • Ding W.X.
        • Ni H.M.
        • Waguri S.
        • Komatsu M.
        Lack of hepatic autophagy promotes severity of liver injury but not steatosis.
        J Hepatol. 2022; 77: 1458-1459
        • Bianco C.
        • Jamialahmadi O.
        • Pelusi S.
        • Baselli G.
        • Dongiovanni P.
        • Zanoni I.
        • et al.
        Non-invasive stratification of hepatocellular carcinoma risk in non-alcoholic fatty liver using polygenic risk scores.
        J Hepatol. 2021; 74: 775-782
        • Singh R.
        • Kaushik S.
        • Wang Y.
        • Xiang Y.
        • Novak I.
        • Komatsu M.
        • et al.
        Autophagy regulates lipid metabolism.
        Nature. 2009; 458: 1131-1135
        • Takamura A.
        • Komatsu M.
        • Hara T.
        • Sakamoto A.
        • Kishi C.
        • Waguri S.
        • et al.
        Autophagy-deficient mice develop multiple liver tumors.
        Genes Dev. 2011; 25: 795-800
        • Ni H.M.
        • Woolbright B.L.
        • Williams J.
        • Copple B.
        • Cui W.
        • Luyendyk J.P.
        • et al.
        Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.
        J Hepatol. 2014; 61: 617-625
        • Karczewski K.J.
        • Francioli L.C.
        • Tiao G.
        • Cummings B.B.
        • Alföldi J.
        • Wang Q.
        • et al.
        The mutational constraint spectrum quantified from variation in 141,456 humans.
        bioRxiv. 2020; 531210
        • Hernandez-Gea V.
        • Ghiassi-Nejad Z.
        • Rozenfeld R.
        • Gordon R.
        • Fiel M.I.
        • Yue Z.
        • et al.
        Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.
        Gastroenterology. 2012; 142: 938-946
        • Fukada H.
        • Yamashina S.
        • Izumi K.
        • Komatsu M.
        • Tanaka K.
        • Ikejima K.
        • et al.
        Suppression of autophagy sensitizes Kupffer cells to endotoxin.
        Hepatol Res. 2012; 42: 1112-1118