Abstract: Background and aims: A variant (p.Arg225Trp) in peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has recently been associated with persistent unexplained hypertransaminasemia and accumulation of C27-BAs, mainly trihy droxycholestanoic acid (THCA). Our aim was to investigate the prevalence of ACOX2 deficiency-induced liver fragility (ADILF) among patients with hypertransaminasemia of unknown origin and their response to ursodeoxycholic acid (UDCA), to identify other inborn errors that could cause this alteration and to elucidate its pathophysiological mechanisms. Method: Serum BA profile was determined by HPLC-MS/MS. Genetic analysis of ACOX2 was performed by exon sequencing. In HuH-7 cells exposed to THCA, viability was determined by MTT, reactive oxygen species (ROS) production by flow cytometry and endoplasmic reticulum (ER) stress by analyzing GRP78 and CHOP levels (RT qPCR and Western Blot), and XBP1-S/XBP1-U ratio (RT-qPCR). The 1000-Genomes database and SIFT and Polyphen scores were used to select ACOX2 variants that were expressed in HuH-7 cells to determine by HPLC-MS/MS its ability to metabolize THCA. Results: Among 33 patients with suspected ADILF from 11 hospitals and 13 relatives, 7 individuals with abnormally high C27-BA levels (>50% of total BAs) were identified. The p.Arg225Trp variant was found in homozygosity in 2 patients and 3 relatives. Moreover, other 2 non-related patients were heterozygous carriers of different alleles, for c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). Impaired expression of ACOX2, but not ACOX3 in the liver of these patients was found (immunohistochemistry). Treatment with UDCA normalized transaminases levels. Culture of HuH-7 liver cells with THCA increased ROS production and the ER stress biomarkers GRP78, CHOP and XBP1-S/XBP1-U ratio, whereas decreased cell viability. THCA-induced toxicity was higher than that of major BAs. Among 14 in silico selected genetic variants, in vitro functional tests identified 6 ACOX2 variants as a potential cause of ADILF. Conclusion: Dysfunctional ACOX2 is found in a substantial proportion of patients with unexplained hypertransaminasemia, suggesting that this disorder of BA metabolism causes enhanced liver fragility, which can be attenuated by UDCA treatment

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