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.
[1]
,[2]
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.[1]
We previously showed that hepatic fat accumulation has a causal role in the progression of liver damage,
[3]
and autophagy is involved in hepatic lipid catabolism in hepatocytes.[4]
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,
[2]
,[5]
,[6]
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.[1]
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[3]
[PRS-HFC]). Conversely, the p.V471A variant was associated with an increased risk of ballooning independently of the steatosis grade.[1]
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.[2]
Table 1Associations between ATG7 p.V471A genotype and liver steatosis grade in subgroup at risk for FLD from the liver biopsy cohort.
| ATG7 genotype | |||
|---|---|---|---|
| Group | Sample size | OR (95% CI) | adj p value |
| T2DM, yes | 506 | 0.89 (0.49–1.59) | 0.70 |
| BMI > 30 | 1,219 | 1.09 (0.72–1.65) | 0.68 |
| PRS-HFC, High | 1,071 | 1.18 (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.
[2]
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.[4]
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.
[7]
In our models, the p.V471A variant resulted in lower ATG7 expression and activity.[1]
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).[1]
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.
[1]
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.
[2]
Concerning non-parenchymal cells, Atg7 deficiency in hepatic stellate cells protected mice from liver fibrosis,[8]
while Atg7 KO was associated with more severe disease in liver endothelial cells and Kupffer cells.[9]
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[5]
,[6]
; 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:
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References
- Rare ATG7 genetic variants predispose patients to severe fatty liver disease.J Hepatol. 2022; 77: 596-606
- Lack of hepatic autophagy promotes severity of liver injury but not steatosis.J Hepatol. 2022; 77: 1458-1459
- Non-invasive stratification of hepatocellular carcinoma risk in non-alcoholic fatty liver using polygenic risk scores.J Hepatol. 2021; 74: 775-782
- Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135
- Autophagy-deficient mice develop multiple liver tumors.Genes Dev. 2011; 25: 795-800
- Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy.J Hepatol. 2014; 61: 617-625
- The mutational constraint spectrum quantified from variation in 141,456 humans.bioRxiv. 2020; 531210
- Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.Gastroenterology. 2012; 142: 938-946
- Suppression of autophagy sensitizes Kupffer cells to endotoxin.Hepatol Res. 2012; 42: 1112-1118
Article info
Publication history
Published online: August 16, 2022
Accepted:
July 28,
2022
Received:
July 21,
2022
Identification
Copyright
© 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
