Abstract: Background and Aims: Inborn errors in bile acid synthesis are rare genetic disorders characterized by accumulation of uncommon bile acid species or their intermediate metabolites, some of them with hepatotoxic effect. The aim of this study was to investigate the case of a young male suffering from persistent hypertransaminasemia of unknown origin, whose clinical features led us to suspect a dysfunction in bile acid metabolism. Results: HPLC-MS/MS analysis of serum and urine revealed very low levels of total major bile acid species in the patient (3 samples) as compared to 5 healthy individuals (serum: 0.06 ± 0.01 vs. 4.68 ± 0.61 ?M; urine: 0.07 ± 0.02 vs. 1.18 ± 0.13 ?M). There was evidence for the presence in serum and urine of the tauroconjugated form of trihydroxycholestanoic acid (THCA), an intermediate of cholic acid synthesis. Its chemical structure was confirmed by Exact Mass determination using HPLC-TOF. To study the genetic bases of this alteration in bile acid metabolism, the exons of all enzymes potentially involved in the accumulation of THCA were amplified by high-fidelity PCR from DNA of the patient. Complete sequencing revealed only an interesting finding: the presence of a homozygotic mutation (c.673C > T, exon 6 of the ACOX2 gene) that results in an amino acid change (p.Arg225Trp) in the peroxisomal enzyme ACOX2, involved in the shortening of the THCA side chain. To investigate the functional consequences, ACOX2 cDNA was obtained from human liver mRNA and cloned. The mutated variant (mutACOX2) was generated by site-directed mutagenesis. Using lentiviral vectors both ACOX2 and mutACOX2 were stably expressed in human hepatoblastoma HepG2 cells. Western blot and immunofluorescence studies revealed that the mutation did not affect the protein size or its subcellular localization. Bile acid synthesis by these cells was studied using THCA as substrate. HPLC-MS/MS analysis showed that the production of cholic acid from THCAwas reduced (?94%) in cells expressing mutACOX2 as compared to ACOX2. Moreover, in cells expressing mutACOX2, but not ACOX2, exposure to THCA (72 h) induced marked oxidative stress (flow cytometry using CellROX®) and impaired cell viability (formazan test). Conclusions: This is the first reported inborn error affecting ACOX2. The result is an impaired bile acid synthesis with accumulation of hepatotoxic metabolites, which could explain the persistent hypertransaminasemia of the patient. This information may be helpful for the selection of the therapy.

Autoría

Descargar referencia