Beneficial effect of ursodeoxycholic acid in patients with acyl-CoA oxidase 2 (ACOX2) deficiency-associated hypertransaminasemia

Background and Aims: A variant (p.Arg225Trp) of peroxisomal acyl-CoA oxidase 2 (ACOX2), involved in bile acid (BA) side-chain shortening, has been associated with unexplained persistent hypertransaminasemia and accumu-lation of C27-BAs, mainly 3α,7α,12α- trihydroxy- 5β- cholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiency-associated hy-pertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), elucidate its pathophysiological mechanism and identify other inborn errors that could cause this alteration.Methods and Results: Among 33 patients with unexplained hypertransami-nasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27-BA levels (>50% of total BAs) were identified by high- performance liquid chromatography-mass spectrometry. The p.Arg225Trp variant was found in homozygosity (exon amplification/sequencing) in two patients and three family members. Two additional nonrelated patients were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with UDCA normalized aminotransferase levels. Incubation of HuH-7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transport-ers, increased reactive oxygen species production (flow cytometry), endo-plasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1- S/ XBP1- Uratio), and BAXα expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was de-creased (tetrazolium salt-based cell viability test). THCA-induced cell toxicity was higher than that of major C24-BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (site-directed mutagen-esis) and expressed in HuH-7 cells. Functional tests to determine their ability to metabolize THCA identified six with the potential to cause ADAH.Conclusions: Dysfunctional ACOX2 has been found in several patients with unexplained hypertransaminasemia. This condition can be accurately identi-fied by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients. This study was supported by the following grants: CIBERehd (EHD15PI05/2016); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI19/00819 and PI20/00189), co-funded by European Regional Development Fund/European Social Fund, “Investing in your future”; “Junta de Castilla y León” (SA074P20); Fundació Marato TV3 (201916–31); AECC Scientific Foundation (2017/2020), Spain; and “Centro Internacional sobre el Envejecimiento” (OLD- HEPAMARKER, 0348_CIE_6_E), Spain. We also acknowledge support from grants PID2019- 111669RBI- 100, PID2020- 115055RB- I00 from Plan Nacional de I+D funded by the “Agencia Estatal de Investigación” (AEI) and the center grant P50AA011999 Southern California Research Center for ALPD and Cirrhosis funded by NIAAA/NIH, as well as support from AGAUR of the “Generalitat de Catalunya” SGR- 2017-1112, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network. Marta Alonso-Peña was the recipient of a predoctoral fellowship from “Ministerio de Educación, Cultura y Deporte” (BOE-A- 2015-9456; FPU-14/00214) and a Mobility Grant for Short Stays from “Ministerio de Ciencia, Innovación y Universidades” (EST17/00186). Ricardo Espinosa-Escudero is the recipient of a predoctoral fellowship from “Junta de Castilla y León” and “Fondo Social Europeo” (EDU/574/2018). The funding sources were not involved in the research design or preparation of the article