MARC1 p.A165T variant is associated with decreased markers of liver damage and increased antioxidant capacity in autoimmune hepatitis


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Between October 2015 and June 2019, we prospectively recruited 313 consecutive non-transplanted adults with AHI at the University of Medicine in Warsaw, Poland. Pure AIH, along with its cholestatic variants, namely AIH-PSC and AIH-PBC, were diagnosed according to current guidelines1 (i.e., they were based on liver histology, the presence of typical antibodies, elevated serum immunoglobulin G concentration, and, in cholestatic variants, imaging studies). The diagnosis of AIH-PSC overlap was based on imaging or histologic features of PSC combined with features (biochemical, serologic and histologic) of AIH. The AIH-PBC diagnosis was established according to the “Paris criteria”23. Any acute or chronic liver disease other than AHI, or its overlap with PSC and PBC, was used as an exclusion criterion.

The study protocol (KB / 128/2015) was approved by the ethics committee of the Warsaw University of Medicine, in accordance with the ethical guidelines of the Helsinki Declaration of 1975 (latest revision, 2013), and written informed consent has been obtained from all participants.

Clinical data

All patients underwent careful clinical examination. Blood samples were taken from fasting subjects. Non-invasive hepatic fibrosis measurements and blood tests were performed in all patients on the same day, i.e. upon enrollment in the study. Hepatic fibrosis was quantified noninvasively using real-time 2D-SWE by Aixplorer (SuperSonic Imagine, Aix-en-Provence, France), as previously described.24, and is presented as LSM. The following serum fibrosis indices were calculated as follows: FIB-4 was calculated according to Sterling et al.25, and APRI was calculated according to Wai et al.26. Liver biopsies were available in 48 (80%) of treatment-naïve patients; BA was not performed in 12 (20%) of treatment-naïve patients due to ascites, coagulopathy, or lack of patient consent. The biopsies were analyzed by a pathologist experienced in liver disease using the Batts and Ludwig27 liver fibrosis and inflammation assessment system. Available clinical data from follow-up were used to examine changes in blood tests from the polymorphisms studied.

Given the differences between pure AIH and its cholestatic variants, we performed genotype-phenotype association tests in the entire patient cohort and separately in subsets of patients with pure AIH and with AIH-. PSC or AIH-PBC. The subgroup of patients with pure AHI was divided according to immunosuppressive therapy (i.e., treatment ≥ 6 months or treatment naïve).

Data at time of diagnosis, clinical outcome and hepatocellular carcinoma

In addition to the data collected at baseline, we analyzed the association between the polymorphisms studied and (1) the age and presence of cirrhosis of the liver at the time of diagnosis, as well as the type of AIH; (2) clinical outcome defined as a combined endpoint: liver transplant (LT) or liver-related death; and (3) hepatocellular carcinoma (HCC). Due to the relatively small number of patients who met the clinical endpoint in the study group (n = 66, 21%), the additional cohort of 30 patients after LT for AIH (57% female, median age at time of diagnosis 33 (range 8–62) years, median LT age 35 (range 12-67) years) was included in this analysis. The diagnosis of HCC was made by imaging tests (CT or MRI) and confirmed by histology, if available.

Genotyping

Genotyping MARC1 (rs2642438), PNPLA3 (rs738409), TM6SF2 (rs58542926), MBOAT7 (rs641738), and HSD17B13 (rs72613567) gene variants was performed at the Saar University Medical Center in Homburg, Germany, by technicians unaware of the patients’ phenotypes. A detailed description of genotyping is provided in Supplementary Text S1 and Table S1.

Mitochondrial damage to hepatocytes related to MARC1 genotype

How? ‘Or’ What MARC1 may impact liver damage is unclear; however, this mitochondrial protein anchored in the signal28 is associated with detoxification reactions29. Thus, we evaluated the presence of markers of mitochondrial damage induced by the oxidative stress of hepatocytes in the serum. The level of mitochondrial decoupling protein 2 (UCP2) (a marker of the inner mitochondrial membrane) and two antioxidant enzymes, thioredoxin reductase 2 (TrxRd2) and superoxide dismutase 2 (SOD2) (markers of the mitochondrial matrix), in the serum of 39 individuals carrying either wild (n = 25) or homozygous (n = 14) MARC1 variant were evaluated. This cohort was randomly selected from the pure AIH group (without AIH overlap with PSC and PBC) treated for at least 6 months. Patients for this analysis were randomly selected from the AIH cohort using SPSS software (IBM Corp. Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp.).

Western blot analysis

The presence of antioxidant enzymes: ubiquitous catalase (CAT) and TrxRd2 and SOD2 located in mitochondria, or mitochondrial proteins like UCP2 in serum, which could be linked to hepatocyte damage, was assessed using specific antibodies, as described in detail in Supplementary Text S2.

Determination of oxidative damage markers in serum

Lipid peroxidation (LPO) in serum was assessed by measuring malondialdehyde (MDA) (a major reactive aldehyde resulting from the peroxidation of biological membranes) using the Abcam lipid peroxidation assay kit (ab118970, Cambridge, UK). Oxidative damage to serum proteins was estimated using the OxyBlot protein oxidation detection kit (S7150, Sigma-Aldrich, MO, USA). A detailed description of the experiments carried out is provided in the supplementary text S2.

Total antioxidant activity of the serum

The concentration of small molecule antioxidants and small protein antioxidants in serum samples was analyzed by measuring the total antioxidant activity (TAA) following ABTS / H2oh2/ HRP method developed by Arnao30 with modifications by Gonzalo-Calvo31 (Additional text S2).

statistical analyzes

Statistical analyzes were performed using SPSS (IBM Corp. Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp.) and GraphPad Prism (GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California United States, http://www.graphpad.com). The consistency of the genotyping results with the Hardy-Weinberg equilibrium (HWE) was tested by exact tests (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). A two-tailed p-value t and Mann-Whitney U tests were used to study normally and non-normally distributed parameters, respectively. Analyzes of variance (ANOVA) and Kruskal-Wallis tests were applied to study the differences between three groups with parameters normally and non-normally distributed, respectively. Wilcoxon’s signed rank test was used to compare the results of the paired blood tests from baseline and follow-up. Associations between genetic variants and follow-up data were tested by logistic regression analyzes. Principal Component Analysis (PCA) was performed with R (2020, R Core Team, Vienna, Austria). The impact of the polymorphisms tested on the clinical endpoint (defined as LT or liver-related death), as well as the diagnosis of HCC, was estimated with Cox regression and Kaplan-Meier survival analysis.

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