Received: 15 November 2021 | Revised: 3 April 2022 | Accepted: 4 April 2022 DOI: 10.1002/hep.32517 ORIGINAL ARTICLE Beneficial effect of ursodeoxycholic acid in patients with acyl-­CoA oxidase 2 (ACOX2) deficiency–­associated hypertransaminasemia Marta Alonso-­Peña1,2 | Ricardo Espinosa-­Escudero1 | Elisa Herraez1,3 | Oscar Briz1,3 | Maria Luisa Cagigal4 | Jesus M. Gonzalez-­Santiago5 | Aida Ortega-­Alonso6 | Conrado Fernandez-­Rodriguez7 | Luis Bujanda3,8 | Marta Calvo Sanchez9 | Delia D´Avola10 | Maria-­Carlota Londoño3,11,12 | Moises Diago13 | Jose C. Fernandez-­Checa3,12,14,15 | Carmen Garcia-­Ruiz3,12,14,15 | Raul J. Andrade3,6 | Frank Lammert16,17 | Jesus Prieto3,10 | Javier Crespo2 | Javier Juamperez18 | Alvaro Diaz-­Gonzalez2 | Maria J. Monte1,3 | 1,3 Jose J. G. Marin 1 Experimental Hepatology and Drug Targeting, Institute for Biomedical Research, University of Salamanca, Salamanca, Spain 2 Gastroenterology and Hepatology Department, Clinical and Translational Research in Digestive Diseases, Valdecilla Research Institute (IDIVAL), Marqués de Valdecilla University Hospital, Santander, Spain 3 Center for the Study of Liver and Gastrointestinal Diseases (CIBEREHD), Carlos III National Institute of Health, Madrid, Spain 4 Pathological Anatomy Service, Hospital Universitario Marqués de Valdecilla, Santander, Spain 5 Department of Gastroenterology and Hepatology, University Hospital of Salamanca, Institute for Biomedical Research, Salamanca, Spain 6 Liver Unit, Gastroenterology Service, Institute of Biomedical Research of Málaga, School of Medicine, University Hospital Virgen de la Victoria, Málaga, Spain 7 Gastroenterology Unit, Fundación Hospital Alcorcón, Rey Juan Carlos University, Madrid, Spain 8 Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country, San Sebastian, Spain 9 Segovia General Hospital, Segovia, Spain 10 Department of Medicine, Clinica Universidad de Navarra and Center for Applied Medical Research, University of Navarra, Pamplona, Spain 11 Liver Unit, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spain 12 Institute of Biomedical Research of Barcelona (IDIBAPS), Barcelona, Spain 13 Valencia University General Hospital, Valencia, Spain 14 Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain 15 Research Center for Alcoholic Liver and Pancreatic Diseases (ALPD) and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California, USA 16 Department of Medicine II, Saarland University Medical Center, Homburg, Germany 17 Health Sciences, Hannover Medical School, Hannover, Germany 18 Pediatric Hepatology and Liver Transplantation Unit, Vall d’Hebron University Hospital, Universitat Autónoma de Barcelona, Barcelona, Spain Abbreviations: ACOX, acyl-­CoA oxidase; ADAH, ACOX2 deficiency-­associated hypertransaminasemia; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CHO, Chinese hamster ovary; DCA, deoxycholic acid; ER, endoplasmic reticulum; GCA, glycocholic acid; HPLC-­MS/MS, high-­performance liquid chromatography-­mass spectrometry; MAF, minor allele frequency; NTCP, Na+-­ taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; ROS, reactive oxygen species; RT-­qPCR, reverse transcription followed by quantitative polymerase chain reaction; TCA, taurocholic acid; THCA, trihydroxycholestanoic acid; UDCA, ursodeoxycholic acid. Marta Alonso-­Peña and Ricardo Espinosa-­Escudero share first authorship and have contributed equally. Maria J. Monte and Jose J.G. Marin are co-­senior authors and have contributed equally. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases. Hepatology. 2022;76:1259–1274.  wileyonlinelibrary.com/journal/hep | 1259 1260 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA Correspondence Jose J. G. Marin, Department of Abstract Physiology and Pharmacology, Campus Miguel de Unamuno E.D. Lab231, Background and Aims: A variant (p.Arg225Trp) of peroxisomal acyl-­CoA 37007- ­Salamanca, Spain. oxidase 2 (ACOX2), involved in bile acid (BA) side-­chain shortening, has been Email: jjgmarin@usal.es associated with unexplained persistent hypertransaminasemia and accumu- Funding information lation of C27-­BAs, mainly 3α,7α,12α-­trihydroxy-­5β-­cholestanoic acid (THCA). This study was supported by the following grants: CIBERehd (EHD15PI05/2016); We aimed to investigate the prevalence of ACOX2 deficiency-­associated hy- Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain pertransaminasemia (ADAH), its response to ursodeoxycholic acid (UDCA), (PI19/00819 and PI20/00189), co-­funded elucidate its pathophysiological mechanism and identify other inborn errors by European Regional Development Fund/European Social Fund, “Investing that could cause this alteration. in your future”; “Junta de Castilla y León” (SA074P20); Fundació Marato TV3 Methods and Results: Among 33 patients with unexplained hypertransami- (201916–­31); AECC Scientific Foundation nasemia from 11 hospitals and 13 of their relatives, seven individuals with (2017/2020), Spain; and “Centro Internacional sobre el Envejecimiento” abnormally high C27-­BA levels (>50% of total BAs) were identified by high-­ (OLD-­HEPAMARKER, 0348_CIE_6_E), Spain. We also acknowledge support performance liquid chromatography-­ mass spectrometry. The p.Arg225Trp from grants PID2019-­111669RBI-­100, variant was found in homozygosity (exon amplification/sequencing) in two PID2020-­115055RB-­I 00 from Plan Nacional de I+D funded by the “Agencia patients and three family members. Two additional nonrelated patients Estatal de Investigación” (AEI) and the center grant P50AA011999 Southern were heterozygous carriers of different alleles: c.673C>T (p.Arg225Trp) and California Research Center for ALPD c.456_459del (p.Thr154fs). In patients with ADAH, impaired liver expression of and Cirrhosis funded by NIAAA/NIH, as well as support from AGAUR of ACOX2, but not ACOX3, was found (immunohistochemistry). Treatment with the “Generalitat de Catalunya” SGR-­ 2017-­1112, European Cooperation in UDCA normalized aminotransferase levels. Incubation of HuH-­7 hepatoma Science & Technology (COST) ACTION cells with THCA, which was efficiently taken up, but not through BA transport- CA17112 Prospective European Drug-­ Induced Liver Injury Network. Marta ers, increased reactive oxygen species production (flow cytometry), endo- Alonso-­Peña was the recipient of a predoctoral fellowship from “Ministerio de plasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1-­S/XBP1-­U Educación, Cultura y Deporte” (BOE-­A -­ ratio), and BAXα expression (reverse transcription followed by quantitative 2015-­9 456; FPU-­14/00214) and a Mobility Grant for Short Stays from “Ministerio polymerase chain reaction and immunoblot), whereas cell viability was de- de Ciencia, Innovación y Universidades” (EST17/00186). Ricardo Espinosa-­ creased (tetrazolium salt-­based cell viability test). THCA-­induced cell toxicity Escudero is the recipient of a predoctoral was higher than that of major C24-­BAs and was not prevented by UDCA. fellowship from “Junta de Castilla y León” and “Fondo Social Europeo” Fourteen predicted ACOX2 variants were generated (site-­directed mutagen- (EDU/574/2018). The funding sources were not involved in the research design esis) and expressed in HuH-­7 cells. Functional tests to determine their ability or preparation of the article 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. I NTRO DUCTI O N its cause is particularly difficult in pediatric patients due to the lack of accompanying clinical manifestations.[2] Hypertransaminasemia is a common condition de- Thus, up to 13% of persistent hypertransaminasemia tected in primary care practice[1] whose most frequent found in children remains cryptogenic.[5] etiologies in adults are nonalcoholic fatty liver dis- Acyl-­CoA oxidase 2 (ACOX2) is a peroxisomal en- ease, viral infections, consumption of toxic substances zyme involved in the shortening of the cholesterol side-­ (mainly alcohol), autoimmune liver disease, celiac dis- chain during bile acid (BA) biosynthesis. Two cases of ease, iron overload, and Wilson disease.[2] Up to 15% ACOX2 deficiency were reported in 2016.[6,7] The first of persistent hypertransaminasemia cases in adults are case, reported at the International Liver Congress-­ idiopathic.[3] Biopsy permits a diagnosis in most (87%) EASL (April 2016; Barcelona, Spain),[6] involved a but not all cases.[4] Information on asymptomatic hyper- 16-­year-­old man with unexplained hypertransaminase- transaminasemia in childhood is scarce, and identifying mia (2-­5 -­fold upper normal limit) and no other associated HEPATOLOGY     | 1261 symptoms. In this individual, a homozygous variant M ATER I A LS A ND M ETHO DS (NM_003500.4:c.673C>T) in ACOX2, resulting in the amino acid change p.Arg225Trp, caused a decrease Reagents and cell lines in enzymatic activity, together with reduced plasma levels of C24-­BAs and the accumulation of C27-­BAs.[8] BA-­related compounds were from Sigma-­ Aldrich The minor allele frequency (MAF) for c.673C>T in the (Merck, Madrid, Spain), except THCA and 7α-­hydroxy-­ Genome Aggregation Database (gnomAD v3.1), includ- 4-­cholesten-­3 -­one (C4), which were from Avanti Polar ing more than 76,000 whole genomes, is 0.03%, which Lipids (Alabaster, AL). Cells used in this study were suggested that this is not a frequent variant found in human embryonic kidney (HEK) 293T, HuH-­7 (human the general population. However, data from the Medical hepatocellular carcinoma), HepG2 and HuH-­6 (human Genome Project obtained recently in a Spanish cohort hepatoblastoma), IHH (immortalized human hepato- of healthy individuals (approximately 2000 genomes) cytes),[16] and stably transfected Chinese hamster showed an MAF of 0.7% for the c.673C>T allele,[9] sug- ovary (CHO),[17] whose origin and culture conditions, gesting that ACOX2 deficiency may not be an extremely as well as the rest of detailed information regarding the rare condition. Indeed, in July 2016, a second case was materials and methods, including statistical analysis, is reported by Vilarinho et al.,[7] which was followed by available in the Supporting Information. a third case identified 2 years later by Ferdinandusse et al.[10] Both children were born from consanguine- ous parents and died during childhood suffering from Human samples severe hepatic and neurological alterations. One of these patients reported carried a homozygous variant Plasma and white blood cells samples were collected (c.207T>A) encoding truncated ACOX2 at codon 69 between 2016 and 2022 in 10 hospitals in Spain and [NM_003500.4:c.207T>A (p.Tyr69Ter)],[7] whereas the one in Germany (Figure S1). The research protocol con- other was homozygous for a four nucleotide deletion formed to ethical guidelines of the 1975 Declaration of (NM_003500.4:c.461_464del), leading to a premature Helsinki. The use of genetic information was approved stop codon (p.Thr154fs).[10] Although elevated serum by the Human Ethical Committees of the University of C27-­BA levels were found, these patients did not pres- Salamanca, Marqués de Valdecilla University Hospital, ent a significant reduction in C24-­BA levels. Both cases and Vall d'Hebron University Hospital (Spain). Informed showed a markedly higher severity in comparison with consent was obtained from patients and relatives enter- that caused by c.673C>T.[8] ing this study. BA accumulation can lead to severe hepatocellular damage, which is often accompanied by inflamma- tory processes.[11] We have previously shown that the Analytical methods most abundant C27-­BAs in the plasma of these pa- tients were 3α,7α,12α-­trihydroxy-­5β-­cholestanoic acid Plasma samples were collected after overnight fasting. (THCA) and its conjugated derivatives, which induces BA[8,18,19] and C4[20,21] concentrations were measured oxidative stress and cell death in cells overexpressing by high-­ performance liquid chromatography-­ mass the c.673C>T ACOX2 variant.[8] Moreover, some BAs spectrometry (HPLC-­MS/MS). Plasma activities of ala- are capable of inducing endoplasmic reticulum (ER) nine aminotransferase (ALT), aspartate aminotrans- stress,[12] releasing Ca2+ from the ER of hepatocytes, ferase (AST), gamma-­ glutamyltranspeptidase and triggering Ca2+- ­dependent apoptosis[12] and activating alkaline phosphatase, as well as total and direct bili- the generation of reactive oxygen species (ROS) by mi- rubin concentrations, were measured using standard tochondria,[13] resulting in cell death,[14] which plays a clinical automatic analyzers. pivotal role in the hypercholanemia-­induced hepatocel- lular damage observed in several liver diseases. To elucidate the prevalence of ACOX2 deficiency-­ Genetic analysis and gene expression associated hypertransaminasemia (ADAH), we an- alyzed a group of 33 such patients from different DNA was obtained from blood samples to amplify the hospitals. We have also explored the mechanisms un- coding sequence of ACOX2 by high-­fidelity polymerase derlying THCA-­induced toxicity. Furthermore, because chain reaction (PCR). The amplified fragments contain- several variants that may affect ACOX2 function have ing both the exons and the exon-­intron boundaries from been predicted,[15] we selected those with the highest at least two reactions of PCR per exon were purified likelihood of impairing ACOX2 function, whose ability by agarose gel electrophoresis. Then, the amplicons to affect BA biosynthesis was then evaluated in vitro. were sequenced in both directions using forward and Finally, and more importantly, we have evaluated the reverse primers as previously described.[8] clinical response of patients with ADAH to ursodeoxy- Total RNA extraction from cells, reverse transcription cholic acid (UDCA) treatment. (RT), and quantitative PCR (qPCR) were performed 1262 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA as previously reported[22] using the appropriate prim- produced in host HEK293T cells[25] and viral titers were ers (Table S1). The results of mRNA abundance of determined by analyzing Enhnaced Green Fluorescent target genes in each sample were normalized using Potein (EGFP)-­positive cells in a FACSCalibur flow cy- hypoxanthine phosphoribosyltransferase 1 (HPRT1) tometer (BD Biosciences, Madrid).[8] HuH-­7 hepatoma expression. target cells were transduced with lentiviral vectors. Overexpression of ACOX2 variants was assessed by RT-­qPCR and immunoblotting. Double limiting-­dilution Histological assessment was performed to obtain monoclonal populations, which were selected according to ACOX2 mRNA and Liver biopsies were processed routinely.[23] They were protein expression, as determined by RT-­qPCR, im- fixed with 10% neutral formalin and embedded in par- munoblot, immunofluorescence, and BA metabolism affin. Serial sections (4 µm) or mirror-­image sections studies. ACOX2 activity was assessed by studying were stained with hematoxylin and eosin or Masson’s the conversion of THCA into cholic acid (CA) in HuH-­7 trichrome (Figure S2). Histopathological findings and cells expressing each variant. Cells were incubated the fibrosis stage were graded by an expert pathologist with 2 µM THCA for 72 h. Biotransformation of THCA using Knodell’s and Scheuer’s scores. into CA was measured by analyzing them in cells and culture medium.[8] Immunohistochemistry, immunoblotting, and immunofluorescence Toxicity and cell stress studies Immunostaining of ACOX2 and ACOX3 in 4-­µm sections Cell viability was determined by the tetrazolium salt-­based of paraffin-­embedded liver biopsies was performed cell viability test using thiazolyl blue tetrazolium bromide using Protein Atlas[24] validated antibodies anti-­ (Sigma-­ Aldrich, Merck). Oxidative stress was deter- ACOX2 (HPA064845, Merck, Madrid) and anti-­ACOX3 mined by flow cytometry using 2′,7′-­dichlorofluorescein (HPA035840, Merck, Madrid), whose specificity was diacetate (Sigma-­Aldrich, Merck). ER stress and apop- tested elsewhere.[10] Immunoblotting analyses of cell tosis were evaluated by analyzing atypical XBP1 splic- lysates were carried out with primary antibodies diluted ing by RT-­qPCR by combining specific primers (Table in phosphate-­ buffered saline-­Tween using blocking S1) for total (XBP1-­ total), short (XBP1-­ S) and long agents as appropriate (Table S2). glyceraldehyde-­3 -­ (XBP1-­U) mRNA. Up-­regulation of genes involved in phosphate dehydrogenase (GAPDH) expression was ER stress (CHOP and GRP78) and apoptosis activa- used as a loading control. tion (BAX and BCL2) was determined by RT-­ qPCR For immunofluorescence studies, cells were (Table S1) and/or immunoblot (Table S2) after incuba- fixed with paraformaldehyde and permeabilized with tion with assayed agents for 24 h. Thapsigargin (2 μM) Triton X-­100. After blocking with fetal bovine serum, and sorafenib (5 µM) were used as a positive controls of cells were incubated for 1 h with anti-­ACOX2 anti- ER stress and apoptosis activation, respectively. body (PA5-­ 50297, Invitrogen, Thermo Fisher) and anticatalase antibody (LF-­ M A0004, Invitrogen, Thermo Fisher). Finally, samples were incubated In silico prediction of dysfunctional with the appropriate secondary antibodies, mouse ACOX2 variants or rabbit anti-­IgG conjugated with Alexa Fluor-­488 or Alexa Fluor-­594 (Invitrogen, Thermo Fisher) and The gnomAD v3.1 (exomes) (https://gnomad.broad​ 4,6- ­diamidino-­2-­phenylindole (Invitrogen, Thermo insti​tute.org) was searched to identify ACOX2 vari- Fisher). Visualization of the labeling was performed ants described in the population that might result in a under a confocal microscope (TCS SP5, Leica, dysfunctional protein. Inclusion criteria for the study of Barcelona). ACOX2 genetic variants were (i) single nucleotide pol- ymorphism; (ii) MAF <1%; and (iii) generation of a mis- sense variant. The selected variants were classified Functional study of ACOX2 variants according to the prediction of the effect of each variant in ACOX2 activity, using SIFT and PolyPhen-­2 algo- The ACOX2 open reading frame (ORF) was amplified rithms. The predicted functional impact score was cal- from human liver RNA and cloned into the pGEM-­T culated as follows: SIFT and PolyPhen-­2 predictions of Easy vector (Promega, Madrid), which was used to the effect of each variant in ACOX2 activity was given generate vectors containing different ACOX2 vari- values from 0 to 2, depending on their predicted ef- ants by site-­ directed mutagenesis (Table S3). The fect by each algorithm (null = 0, probable = 1, or highly mutant ORF was then transferred to lentiviral vectors probable = 2). The sum of both scores resulted in a containing a V5-­tag.[8] Recombinant lentiviruses were scale from 0 to 4 of which 0 indicated the variant was HEPATOLOGY     | 1263 most likely benign and 4 indicated a high probability of suggested a preliminary diagnosis of seronegative auto- deleterious effects. immune hepatitis. Accordingly, the patient received im- munosuppressive drugs for 10 years, but biochemical remission was not achieved. Lack of response and per- Transport assays sistent hypertransaminasemia during this time prompted new clinical evaluation. Again, all common causes of BA uptake by HuH-­7 cells and transporter-­mediated liver and biliary tract disorder were ruled out. A new liver uptake by CHO cells was determined as previously biopsy (2019) showed no additional information (Figure described.[26] HuH-­7 and CHO cells, either wild-­type S2). Finally, as the first episode of hypertransaminasemia or stably expressing Na+-­taurocholate cotransporting had been recorded after treating a testicular torsion with polypepetide (NTCP), organic anion transporting poly- nonsteroidal anti-­inflammatory drugs, which resembled peptide-­1B1 (OATP1B1), or OATP1B3,[17,27–­29] were in- the first case of ADAH previously described,[6,8] following cubated with uptake medium containing 50 µM THCA, an empiric approach, UDCA therapy was started. After glycocholic acid (GCA), or CA with or without 250 µM 3 weeks, plasma aminotransferases reverted to normal taurocholic acid (TCA) for 60 min. Uptake was stopped (Table S4), and fatigue symptoms improved. Further anal- by rinsing the wells twice with cold uptake medium and ysis revealed that the patient showed an increased pro- twice with cold PBS. Cells were lysed with ultra-­pure portion of C27-­BAs (Figure 1, Table 1) and the presence water containing 5 µM of taurochenodeoxycholic acid in homozygosis of the previously described[8] ACOX2 (TCDA; internal standard), and BA concentrations were variant c.673C>T (p.Arg225Trp) (Figure 1). The study of determined by HPLC-­MS/MS as described above. his family revealed that his brother, mother, and uncle car- ried this variant in homozygosis and accumulated C27-­ BAs in serum (Figure 1, Table 1). Interestingly, all had R ESULTS undergone a cholecystectomy because of complicated cholelithiasis. Biochemical analysis of the uncle revealed Patients with idiopathic hypertransaminasemia (ALT/AST: 131/68 U/l), which led hypertransaminasemia his physician to prescribe him UDCA therapy. This re- stored his aminotransferase levels to normal values after Thirty-­ three patients with persistent hypertransamina- 3 weeks (ALT/AST: 27/19 U/l). Serum aminotransferase semia of unknown origin entered this study after ruling levels were still low after 6 months of treatment (ALT/AST: out common causes of elevated serum aminotrans- 17/20 U/l), even though the abnormal proportion of C27-­ ferases (viral hepatitis, alcoholic or metabolic liver dam- BAs was not corrected (Figure 1, Table 1). In contrast, age, autoimmune hepatitis, hemochromatosis, Wilson's the father, who was heterozygous for the same variant, disease, alpha 1 antitrypsin deficiency, and obstructive and the aunt, who did not carry any ACOX2 variant, were cholestasis). Fourteen of these subjects showed hyper- negative for C27-­BA accumulation (Figure 1, Table S8). cholanemia (≥10 µM), suggesting some degree of chol- estasis. Among the rest of normocholanemic individuals, a C27-­BAs/C24-­BAs ratio >1 was found in four subjects, Case 2 who were considered as potential ADAH cases. Thirteen relatives from these patients were also analyzed. Among A 17-­year-­old boy with persistent and unexplained hy- them, three individuals had enhanced C27-­BAs levels pertransaminasemia was first seen in the Hepatology together with genetic data consistent with dysfunctional Unit of Marqués de Valdecilla University Hospital in ACOX2 (Figure 1). Plasma BA profiles of the individu- 2020. He had previously been diagnosed with growth als with confirmed ADAH are shown in Table 1. No sig- hormone deficiency, treated with somatropin, and nificant alteration was detected in some cases in which followed up by his pediatrician in another center. serum C4 levels were measured (Table 1). Hypertransaminasemia had been recorded since his first year of life (Table S5). Usual and unusual causes of persisting hypertransaminasemia were discarded, Case 1 including lysosomal acid lipase deficiency. Abdominal ultrasound and MRCP showed no relevant findings. A 17-­year-­old boy was first seen in 2010 in the Hepatology Liver biopsy showed no remarkable signs: slight si- Unit of Marqués de Valdecilla University Hospital nusoidal dilatation and minimal central vein fibrosis (Santander, Spain) due to fatigue symptoms and unex- (Figure S2). The analysis of plasma BAs revealed an plained hypertransaminasemia (Table S4). Abdominal increased proportion of C27-­BAs (Figure 1, Table 1). ultrasound showed normal liver and biliary tract. A liver The genetic studies showed that he was homozy- biopsy (2010) revealed the presence of a single portal gous for the ACOX2 variant c.673C>T (p.Arg225Trp) fibrous enlargement with focal porto-­portal bridging fibro- (Figure 1). UDCA treatment was initiated, achieving sis and minimal portal inflammatory component, which normalization of aminotransferases (Table S5) and 1264 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA F I G U R E 1 Genealogical tree of families with members showing acyl-­CoA oxidase (ACOX) 2 deficiency-­associated hypertransaminasemia (ADAH) (left panels) and the proportion of plasma concentrations of endogenous bile acids (BAs) with short side-­ chain (C24-­BAs) and immature BAs with unshorten side-­chain (C27-­BAs) (right panels). In individuals treated with ursodeoxycholic acid (UDCA; 12 or 15 mg/kg/day), this BA and its conjugates were not considered for calculating plasma endogenous C24-­BA concentrations. reduction in the proportion of C27-­BAs (Figure 1) after Case 3 3 weeks. Nevertheless, the follow-­up for 6 months of treatment revealed abnormal abundance of serum A 15-­year-­old boy in follow-­up at Santa Creu i Sant C27-­BAs (Figure 1, Table 1), despite the correction of Pau Hospital (Barcelona, Spain) since the first years serum aminotransferase levels was maintained (Table of life due to abdominal distension, steatorrhea, and S5). The analysis of his family showed that his younger hypertransaminasemia was first seen in the Pediatric brother was homozygous for wild-­ t ype ACOX2, Hepatology and Liver Transplantation Unit from Vall whereas the parents were heterozygotic carriers for d’Hebron Hospital (Barcelona, Spain) in 2017. All clini- c.673C>T (p.Arg225Trp) (Figure 1). The plasma BA cal studies performed were negative, including the profiles in the family members analyzed were normal. analysis of classic inborn errors of BA biosynthesis. An C24-­BA plasma levels were within the normal range in abdominal ultrasound revealed discrete hepatic hyper- the mother and brother, whereas the father presented echogenicity, and a liver biopsy showed slight fibrosis hypercholanemia (22 µM total BAs) with general en- (Figure S2). UDCA was empirically administered before hanced levels of all BA molecular species but without ADAH was diagnosed. The treatment resulted in the an elevated proportion of C27-­BAs versus C24-­BAs normalization of plasma aminotransferases (Table S6) (Figure 1; Table S9). and symptoms relief. Then, BA profiling (Table 1) and TA B L E 1 Bile acid species in serum collected from patients with acyl-­CoA oxidase (ACOX) 2 deficiency–­associated hypertransaminasemia (ADAH) Controls Case 1 Uncle case 1 Case 2 Case 3 Case 4 UDCA treatment (months) None 5 None 0.75 6 None 0.75 3 6 41 47 None 0.75 5 11 HEPATOLOGY 7α- ­OH- ­4 -­cholesten-­3 -­one (C4) (nM) 17 ± 3 16 12 11 38 9 16 6 11 ND ND ND ND ND ND Endogenous C24-­bile acids (nM) 2290 ± 500 980 610 1137 1895 620 13,545 295 1336 960 600 703 1454 656 649 Endogenous C27-­bile acids (nM) 20 ± 3 1304 1334 2201 6122 791 5581 153 5014 2315 795 3245 2092 307 512 C27-­bile acids (%) 0.9 ± 0.1 57.1 68.6 65.9 76.4 56.1 29.2 34.1 79.0 70.7 57.0 82.2 59.0 31.9 44.1 C24-­bile acid species (nM) Glyco-­cholic 117 ± 40 43 66 152 615 18 607 13 227 163 30 102 134 27 15 Glyco-­chenodeoxycholic 421 ± 90 256 201 662 1018 40 2289 84 676 461 129 284 664 460 67 Glyco-­deoxycholic 180 ± 44 27 2 74 31 11 450 19 144 5 8 101 71 2 33 Glyco-­lithocholic 9±2 4 <1 46 7 2 4 22 14 1 5 1 6 1 18 Glyco-­ursodeoxycholic 51 ± 17 5287* 6 7998* 4232* 16 13,065* 1667* 6264* 9949* 2524* 107 9281* 3305* 1477* Tauro- ­cholic 18 ± 7 1 5 11 57 <1 24 <1 21 1 5 14 11 1 4 Tauro-­chenodeoxycholic 53 ± 11 14 22 50 88 4 60 2 75 44 12 57 83 29 7 Tauro- ­deoxycholic 23 ± 6 3 1 9 7 1 6 1 13 2 1 26 8 1 2 Tauro-­lithocholic 1±1 <1 <1 3 2 <1 <1 <1 2 <1 <1 <1 1 <1 1 Tauro-­ursodeoxycholic 2±1 97* <1 117* 201* <1 189* 15* 230* 235* 63* 7 263* 64* 33* Tauro-­sulfo-­lithocholic 103 ± 15 3 4 78 3 8 18 10 72 31 205 14 11 1 189 Cholic 403 ± 92 122 276 5 47 504 6546 63 27 93 56 6 33 18 153 Chenodeoxycholic 330 ± 180 445 14 20 11 6 2755 32 19 140 119 41 311 110 46 Deoxycholic 417 ± 124 59 4 13 5 7 781 34 29 8 16 45 114 4 67 Lithocholic 93 ± 27 1 1 14 4 1 5 17 16 1 13 9 10 1 48 Ursodeoxycholic 65 ± 21 6737* 2 7645* 96* 3 105,873* 1276* 223* 3197* 2725* 23 2812* 2555* 1296* C27-­bile acid species (nM) Glyco-­trihydroxycholestanoic <1 118 156 189 763 38 1133 24 521 290 59 153 167 27 54 Glyco-­dihydroxycholestanoic <1 15 39 127 117 19 79 8 61 30 7 18 33 <1 3 Tauro-­trihydroxycholestanoic 16 ± 3 706 902 1348 4492 553 3199 71 3443 1575 597 2125 1432 194 291 Tauro-­dihydroxycholestanoic <1 87 204 472 708 164 364 14 959 333 77 936 438 69 118 Trihydroxycholestanoic 4±1 379 32 63 42 17 806 36 30 87 55 14 21 16 46 Note: Control samples were collected from 18 healthy individuals. UDCA-­associated species were marked with an asterisk (*) when the patient was treated with UDCA (12 or 15 mg/kg/day). Abbreviations: ND, not determined; UDCA, ursodeoxycholic acid.     | 1265 1266 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA directed genetic studies were performed. The patient aortic insufficiency) with slight developmental delay showed an increased proportion of plasma C27-­BAs and oscillating hypertransaminasemia (Table S7). He (Figure 1) and two heterozygous variants, presum- was referred to the Pediatric Hepatology and Liver ably on two different alleles of ACOX2, the c.673C>T Transplantation Unit from Vall d’Hebron Hospital, variant and a deletion of 4 nucleotides (c.456_459del) where common causes of liver damage were ruled (Figure 1). This was predicted to generate the same out. Abdominal ultrasound showed slight hepatic hy- protein change (p.Thr154fs) that had been previously perechogenicity. Liver biopsy presented subtle po- described for a similar variant identified in ACOX2 by lygonal morphology of the hepatocytes with no other Ferdinandusse et al.[10] Liver function tests and anal- remarkable findings (Figure S2). Due to the mul- ysis of BA profile after another 6 months of UDCA tiorgan affectation, a complete exome sequencing treatment revealed maintained normalization of ami- study was indicated. This showed the same double notransferases besides a persistently altered BA pro- heterozygous variant of ACOX2 as in Case 3; i.e., file (Figure 1, Table 1, and Table S6). The analysis of variants c.673C>T (p.Arg225Trp) and c.456_459del the proband’s family showed that the younger brother (p.Thr154fs) (Figure 1). Plasma BA profiling (Table 1) was homozygous for wild-­type ACOX2, whereas the confirmed enhanced C27-­BA proportion (Figure 1). father was heterozygous for c.456_459del (p.Thr154fs) Diagnosis of ADAH prompted UDCA treatment, re- and the mother showed the c.673C>T (p.Arg225Trp) sulting in the normalization of aminotransferase lev- variant in heterozygosis (Figure 1). Their plasma BA els (Table S7) and a partial reduction in the proportion profiles were normal (Figure 1, Table S10), with C27-­ of C27-­BAs in plasma (Figure 1, Table 1). The anal- BAs and C24-­BAs within normal ranges. ysis of the proband’s family members showed that the father carried the c.456_459del (p.Thr154fs) variant in heterozygosis and the mother presented Case 4 the c.673C>T (p.Arg225Trp) variant in heterozygo- sis (Figure 1). In both parents, plasma C27-­BA and A 7-­year-­old boy presented a history of congenital heart C24-­BA levels were within normal ranges (Figure 1; disease (mild mitral stenosis, and moderate-­severe Table S11). F I G U R E 2 Immunohistochemistry analysis of acyl-­CoA oxidase (ACOX) 2 expression in liver biopsies collected from patients with ACOX2 deficiency–­associated hypertransaminasemia (ADAH). Two samples from patients with hypertransaminasemia but not ADAH, namely, NAFLD F1 and alcoholic cirrhosis used as Control 1 (A) and Control 2 (B), respectively. When expressed, ACOX2 was detected as intense granular staining inside the hepatocytes. Negative staining for ACOX2 was seen in liver biopsies from probands of Case 1 (C), Case 2 (D), Case 3 (E), and Case 4 (F). All images were obtained at 20× magnification. Scale bar indicates 50 µm. HEPATOLOGY     | 1267 Immunohistochemistry studies not reduced by UDCA (Figure 4P), whereas BCL2 was scarcely expressed by HuH-­7 cells (Figure 4Q). Immunohistochemistry of liver biopsies showed a com- Consistently, THCA-­induced cell death was not inhib- plete absence of ACOX2 staining in patients with ADAH ited by UDCA (Figure 4R). (Figure 2). In contrast, staining was positive in a series of biopsy samples collected from adult patients with different liver diseases (data not shown), two of which Potentially harmful ACOX2 variants have been included in Figure 2 as controls. In contrast, immunohistochemistry for ACOX3, another acyl-­CoA The search for variants that could cause ADAH, car- oxidase that does not metabolize C27-­BA,[10] showed ried out in the databases, led to the selection of 46 positive signal (with different intensity) in hepatocytes variants, of which most (32 variants) had an extremely in all cases (Figure S3). low MAF. Hence, a maximum contribution of 1.56% of all predicted cases of dysfunctional ACOX2 could be expected. Accordingly, this set was discarded from C27-­BA–­induced toxicity the study and attention was focused on the functional analysis of the remaining 14 variants (Table 2). This To analyze the cytotoxic effects of THCA, the main specific set included the c.673C>T (p.Arg225Trp) vari- unconjugated C27-­BA synthesized in ADAH cases, ant initially identified (ACOX2-­V1). According to SIFT we first analyzed the ability of THCA to enter liver and PolyPhen-­2 predictions, only 11 of these variants cells. CA and GCA uptake by HuH-­7 cells was in- are expected to have a functional impact on ACOX2 hibited by TCA, which was also taken up by these enzymatic activity. These predictions were further vali- cells. THCA uptake was higher and not affected dated by measuring the ability of the variants, when by TCA (Figure 3A). CA, GCA, and TCA uptake by expressed in HuH-­7 cells (Figure 5A,B), to enhance CHO cells was markedly enhanced by NTCP expres- THCA biotransformation into CA. Monoclonal HuH-­7 sion (Figure 3B) but very moderately by OATP1B1 cells stably overexpressing ACOX2-­W T or ACOX2-­V1 (Figure 3C) and OATP1B3 (Figure 3D) expression. were used as positive and negative controls, respec- In all circumstances, a substantial THCA uptake was tively (Figure 5C,D). The activity of variants V11 and found, which was only slightly increased by NTCP ex- V14 was similar to that of ACOX2-­W T, suggesting that pression and was not inhibited by TCA; THCA abil- these variants do not have a functional impact on en- ity to induce oxidative stress was tested in liver cells. zyme activity, as was predicted by in silico analysis. In THCA-­induced ROS production was higher than that contrast, variants V5 and V8 showed a moderately de- induced by C24-­BAs, such as CA, chenodeoxycholic creased activity, whereas variants V2, V4, V6, and V10 acid (CDCA), and deoxycholic acid (DCA) in HuH-­7 were significantly less functional (Figure 5E). and HuH-­ 6 cells, whereas HepG2 and IHH cells showed resistance to BA-­ induced oxidative stress (Figures 4A– ­D). D I SCUSS I O N To study the effect on ER stress, cells sensitive (HuH-­7) and resistant (HepG2) to BA-­induced oxidative This study identified seven individuals from four un- stress were selected. THCA induced a strong effect, related families carrying ACOX2 variants involved in which in most tests was stronger than that caused by ADAH. Among them, five individuals showed persistent C24-­BAs (Figure 4E–­K). oscillating hypertransaminasemia. Three patients were The reduction in viability of HepG2 and HuH-­ 7 homozygous carriers of c.673C>T (p.Arg225Trp), the cells after incubation with THCA was concentration-­ same variant found in the first ADAH case reported,[8] dependent in both cell lines, with HepG2 being less whereas the other two carried heterozygous c.673C>T sensitive than HuH-­ 7 to THCA-­ induced cell death and c.456_459del ACOX2 variants in different alleles. (Figure 4L,M). To confirm the link between THCA-­ Interestingly, the latter generates p.Thr154fs, which re- toxicity and the presence of the p.Arg225Trp variant sults in a premature stop codon and a truncated protein. in ACOX2, monoclonal HuH-­ 7 cell sublines overex- This change is also caused by another similar deletion pressing wild-­type ACOX2 (ACOX2-­W T) or the p.Ar- (c.461_464del) in ACOX2 mRNA described.[10] In con- g225Trp mutated variant (ACOX2-­V1) were generated trast to our patients, the subject of that study carried the (Figure 5). ACOX2-­ W T overexpression partly pro- deletion in homozygosity and presented a more severe tected cells from THCA-­induced cytotoxicity, whereas condition, with neurological symptoms and affection of this C27-­BA caused a similar reduction in cell viabil- lung, liver, heart, and muscle functions, and only sur- ity in control (Mock) and ACOX2-­V1 cells (Figure 4N). vived 6 months.[10] The presence of other congenital THCA-­ induced oxidative stress was not prevented alterations responsible for the multiorgan dysfunction by coincubation with UDCA (Figure 4O). Moreover, of this case was not ruled out. The clinical conditions THCA markedly upregulated BAX mRNA, which was of the six patients with ADAH we have described so far 1268 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA F I G U R E 3 Uptake of glycocholic (GCA), taurocholic acid (TCA), cholic (CA), and trihydroxycholestanoic (THCA) acid by wild-­t ype HuH-­7 hepatoma cells (A) and Chinese hamster ovary (CHO) cells (Control) or CHO cells stably expressing NTCP (B), OATP1B1 (C), or OATP1B3 (D). Cells were incubated with 50 µM GCA, CA, or THCA, without or with 250 µM TCA for 1 h. Bile acid (BA) concentrations in cell lysates were determined by high-­performance liquid chromatography-­mass spectrometry (HPLC-­MS/MS). Values are mean ± SD from eight measurements per condition, carried out in four different cultures. *p < 0.05 by comparing with and without transporter expression; † p < 0.05 by comparing uptake in the presence and the absence of TCA, using paired t test. HEPATOLOGY     | 1269 F I G U R E 4 Effect of trihydroxycholestanoic (THCA), cholic (CA), chenodeoxycholic (CDCA), and deoxycholic (DCA) acids on the generation of reactive oxygen species (ROS) in HepG2 (A), HuH-­7 (B), IHH (C), and HuH-­6 (D) cells. Effect of THCA, CA, CDCA and DCA on the expression (mRNA) of endoplasmic reticulum (ER) stress markers: GRP78 (E,F); XBP1 (unspliced form XBP1-­U and spliced form XBP1-­S) (G,H); and CHOP (I,J). Changes in the expression of GRP78 and CHOP were confirmed by immunoblot (K). ER stress induced by 2 μM thapsigargin (TG) was used as a positive control. Effect of THCA on the viability of HepG2 (L), and HuH-­7 (M) cells. Effect of THCA on the viability of monoclonal HuH-­7 cells overexpressing wild-­type acyl-­CoA oxidase (ACOX) 2 (ACOX2-­WT), the p.Arg225Trp variant of ACOX2 (ACOX2-­V1) or transduced with empty lentiviral vectors (Mock) (N). Effect of ursodeoxycholic acid (UDCA) on THCA-­induced oxidative stress (O), BAX (P), and BCL2 (Q) mRNA levels in wild-­type HuH-­7 cells and on cell viability reduction (R) in HuH-­7 without (Mock) or with stable expression of ACOX2-­WT or ACOX2-­V1. Values (mean ± SD from 6 to 12 determinations performed in 3–­6 different cultures) are expressed as the percentage of data determined in cells treated with the vehicle alone (Control). *p < 0.05, compared to Control; †p < 0.05, comparing to Mock by paired t test. 1270 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA F I G U R E 5 Acyl-­CoA oxidase (ACOX) 2 mRNA expression determined by reverse transcription followed by quantitative polymerase chain reaction (RT-­qPCR) in HuH-­7 control cells transduced with empty lentiviral vector (Mock) and different polyclonal subpopulations overexpressing wild-­type (WT) or ACOX2 variants (V1-­V14) (A). ACOX2 protein expression determined by Western-­Blot using anti-­V5 antibody in lysates from Mock cells and polyclonal cells overexpressing ACOX2 variants from V2 to V14 (B), and monoclonal cells overexpressing WT or V1 variant. GAPDH was used as loading control (C). Representative immunofluorescence images of HuH-­7 cells without (Mock) or with overexpression of ACOX2 WT or V1 variant, showing the subcellular localization of peroxisomal catalase (red), ACOX2 (green, using an anti-­ACOX2 antibody) and its colocalization (merged, yellow). Nuclei were labeled with 4,6-­diamidino-­2-­phenylindole (blue) (D). Change in the ability of HuH-­7 cells expressing ACOX2 variants to biotransform trihydroxycholestanoic acid (THCA) into cholic acid (CA) (E). Six weeks after transduction, the cells were incubated with 2 μM THCA for 72 h. CA concentrations in the culture medium plus cell lysate were determined by high-­performance liquid chromatography-­mass spectrometry (HPLC-­MS/MS). Monoclonal sublines stably expressing ACOX2 WT or V1 were used as positive (+) and negative (-­) controls, respectively. Values are mean ± SD from at least six determinations performed in three different cultures. *p < 0.05, compared with control (Mock) using the Bonferroni multiple comparison test. markedly differ from that case, showing severe symp- variability, as in the case of alpha-­ methylacyl-­ CoA toms unrelated to liver function only in Case 4. Besides racemase (AMACR)[30] and bile acid-­CoA:amino acid hypertransaminasemia, no symptoms were observed N-­acyltransferase (BAAT)[31] deficiencies. in several of our patients, as in the first reported case As a standard feature, all individuals with ADAH of ADAH.[8] Other defects in the peroxisomal pathway showed an increased proportion of C27-­BAs (56%–­86% of BA biosynthesis also show great interindividual of total plasma BAs) (Figure 1, Table 1). Interestingly, HEPATOLOGY     | 1271 T A B L E 2 Definition, nomenclature, and prediction of the functional consequence of the most frequent missense mutations in the ORF of acyl-­CoA oxidase 2 (ACOX2) mRNA Nucleotide Amino acid Functional Name MAF Alleles ID change change impact V1 A = 0.030% G, A rs150832314 673C>T Arg225Trp 4 V2 A = 0.054% G, A rs143133121 1840C>T Arg614Cys 3 V3 T = 0.010% C, T rs529617348 674G>A Arg225Gln 3 V4 G = 0.011% C, G rs142358437 1216G>C Glu406Gln 4 V5 C = 0.001% T, C rs578023477 790A>G Arg264Gly 4 V6 G = 0.301% A, G rs114465932 713T>C Ile238Thr 2 V7 T = 0.035% C, T rs112279882 946G>A Ala316Thr 3 V8 A = 0.026% G, A rs151184272 1720C>T Arg574Cys 2 V9 T = 0.032% C, T rs143508132 850G>A Gly284Ser 2 V10 A = 0.0004% C, A rs143508132 850G>T Gly284Cys 2 V11 A = 0.010% T, A rs550491341 1087A>T Ser363Cys 1 V12 C = 0.058% G, C rs143228600 1582C>G Gln528Glu 0 V13 T = 0.002% C, T rs191136907 1984G>A Glu662Lys 0 V14 T = 0.024% C, T rs149888394 841G>A Val281Ile 0 Note: Data, updated in September 2021, were obtained from the Genome Aggregation Database (gnomAD v3.1, Exomes). The nucleotide position refers to the open reading frame of the wild-­t ype sequence NM_003500 and the amino acid position is based on the protein NP_003491. The predicted functional impact score was calculated as follows: SIFT and PolyPhen-­2 predictions of the effect of each variant in ACOX2 activity was given values from 0 to 2, depending on their predicted effect by each algorithm (null = 0, probable = 1, or highly probable = 2). The sum of both scores resulted in a scale from 0 to 4 in which 0 indicates that the variant is probably benign, whereas 4 indicates a high probability of deleterious effects. Abbreviations: MAF, minor allele frequency; V, variant. C24-­BA biosynthesis was reduced but not completely systemic (e.g., change in BA pool composition, which is abolished (Table 1, Tables S8–­S11). This finding points enriched in UDCA-­related species, more polar and less to a certain remnant ACOX2 activity or the existence toxic than C27-­BAs) levels. Another interesting finding of a yet unknown mechanism capable of overcoming associated with UDCA treatment is that, as expected, ACOX2 deficiency, the only recognized enzyme able to based on its poor ability to activate FXR, administered metabolize C27-­BAs.[7,8,10] In this sense, Shefer et al. UDCA cannot inhibit de novo synthesis of BAs and previously proposed, in 1976,[32] an alternative pathway hence cannot inhibit C27-­BAs production, which rec- in both humans and rats for BA β-­oxidation, named the ommends additional pharmacological intervention for a 25-­hydroxylation pathway, that does not require the better long-­term management of patients with ADAH. participation of peroxisomes. The fact that gallbladder disease was reported in UDCA has been used for the treatment of several some adult individuals with ADAH prompted the sus- cholestatic liver diseases[33] due to its multiple benefi- picion of a possible link between C27-­BA accumulation cial effects, which include strong choleretic activity and and cholelithiasis. The low number of cases analyzed protection of hepatocytes and cholangiocytes from the here and their occurrence in only one family preclude toxic effects of more hydrophobic BA species. This has robust conclusions in this respect. been suggested to occur owing to its antioxidant, anti- Although p.Arg225Trp variant affected just one apoptotic, and immunomodulatory properties,[34] which amino acid, the staining of ACOX2 in liver biopsies was has justified its use as an anti-­inflammatory drug in negative in all affected patients. The protein structure treating several liver diseases.[35] analysis revealed that the amino acid change might be Here, we report the positive effects of UDCA in the relevant for ACOX2 dimerization.[8] Thus, the variant treatment of patients with ADAH, reverting aminotrans- could hinder dimerization or result in altered dimers ferases to normal levels and ameliorating accompa- with a greater probability of being degraded by the pro- nying symptoms, after 3 weeks of UDCA treatment, teasome. Recently, some studies have also described despite the lack of correction in the abnormal proportion the absence of protein expression due to the presence of C27-­BAs (Figure 1). Results from our in vitro studies of missense gene variants.[36,37] The mechanism ac- suggest that UDCA-­ mediated prevention of harmful counting for enhanced mRNA degradation in these THCA-­induced effects does not mainly occur at hepato- cases is still poorly understood. cellular level but rather owing to mechanisms acting at In contrast with conjugated and unconjugated forms the organ (e.g., hepatobiliary washing associated with of CA, THCA is taken up by a process markedly indepen- marked UDCA-­induced stimulation of bile formation) or dent of NTCP, OATP1B1, and OATP1B3. Regarding the 1272 |    RESPONSE OF ACOX2 DEFICIENCY TO UDCA mechanisms accounting for C27-­BAs noxious effects, Javier Juamperez, Marta Alonso-­ Peña, Ricardo our results show that THCA is more toxic than DCA and Espinosa-­ Escudero, Alvaro Diaz-­ Gonzalez, Javier CDCA, which are primary mediators of hepatotoxicity Crespo, Maria J. Monte, Jose J. G. Marin. Experiments in situations of C24-­ BA accumulation.[12,38] C27-­BA-­ and procedures: Marta Alonso-­Peña, Ricardo Espinosa-­ induced hepatocellular toxicity can be partly due to im- Escudero, Elisa Herraez, Oscar Briz, Maria J. Monte. paired mitochondrial respiratory function and increased Interpretation and discussion: Marta Alonso-­ Peña, ROS production.[39] Although intrahepatic concentrations Ricardo Espinosa-­Escudero, Elisa Herraez, Oscar Briz, of C27-­BAs in patients with ADAH may be variable and Maria Luisa Cagigal, Jesus M. Gonzalez-­Santiago, Aida lower than those used in our in vitro experiments, chronic Ortega-­Alonso, Conrado Fernandez Rodriguez, Luis exposure to C27-­BAs and the concurrence of other del- Bujanda, Marta Calvo Sanchez, Delia D´Avola, Maria-­ eterious factors may promote hepatocellular vulnerability Carlota Londoño, Moises Diago, Jose C. Fernandez-­ and eventually result in liver damage. Checa, Carmen Garcia-­ Ruiz, Raul J. Andrade, Frank Finally, we have identified six ACOX2 variants that, Lammert, Jesus Prieto, Javier Crespo, Javier Juamperez, when expressed in HuH-­7 cells, show impaired ability Alvaro Diaz-­Gonzalez, Maria J. Monte, Jose J. G. Marin. to enhance THCA biotransformation into CA and there- Writing of the article: Marta Alonso-­Peña, Maria J. Monte, fore can be considered candidates to cause ADAH. Jose J. G. Marin. Revision of the article: Marta Alonso-­ This in vitro study has some limitations, such as mea- Peña, Ricardo Espinosa-­Escudero, Elisa Herraez, Oscar suring the final product of the biochemical pathway, Briz, Maria Luisa Cagigal, Jesus M. Gonzalez-­Santiago, CA, instead of the direct product of ACOX2 activity, the Aida Ortega-­Alonso, Conrado Fernandez Rodriguez, Luis 24-­enoyl-­CoA derivative of THCA. This may limit the ca- Bujanda, Marta Calvo Sanchez, Delia D´Avola, Maria-­ pacity to discriminate the activity of the overexpressed Carlota Londoño, Moises Diago, Jose C. Fernandez-­ ACOX2 variant due to the necessary action of enzymes Checa, Carmen Garcia-­ Ruiz, Raul J. Andrade, Frank downstream of the pathway, i.e., D-­bifunctional protein Lammert, Jesus Prieto, Javier Crespo, Javier Juamperez, (DBP) and sterol carrier protein x (SCPx). Thus, even Alvaro Diaz-­Gonzalez, Maria J. Monte, Jose J. G. Marin. though our results add a valuable piece of evidence about the predicted negative impact of ACOX2 variants, ACKNOWLEDGMENTS their deleterious consequences cannot be confirmed The authors thank Maria Dolores Herrera Cisneros, until the diagnosis of additional ADAH individuals car- Yolanda Palacin Viaña, Sara Arias, Andrea Fernandez rying these variants could be made. Therefore, more Rodriguez, and David Merino for technical support. extensive genetic studies in subjects presenting idio- The English revision of the manuscript by Emma Keck pathic hypertransaminasemia and other hepatic pa- is sincerely appreciated. The authors also would like thologies of unknown origin, should be encouraged to kindly thank the participants of this study who have to gather accurate data on ADAH prevalence. Still, it made this project possible. is foreseeable that more cases of ADAH will appear in the future among patients with unexplained hyper- CONFLICTS OF INTEREST transaminasemia carrying ACOX2-­V1 or other variants Dr. Bujanda consults for Ikan Biotech. with predicted functional impact and high MAF, such as ACOX2-­V2 and ACOX2-­V6, which are expected to be ORCID present in approximately 1 and 5 out of 2000 exomes, Marta Alonso-­Peña https://orcid. respectively. org/0000-0003-0934-2202 Finally, the two most relevant clinical contributions Jesus M. Gonzalez-­Santiago https://orcid. of the present study are that a noninvasive and effec- org/0000-0003-4667-4492 tive diagnostic strategy based on plasma BA profiling Maria-­Carlota Londoño https://orcid. and ACOX2 sequencing can accurately identify pa- org/0000-0002-6533-1586 tients with ADAH and that their liver damage can be Javier Crespo https://orcid. efficiently attenuated by UDCA treatment. org/0000-0001-8248-0172 Maria J. Monte https://orcid.org/0000-0003-1844-7428 AUTHOR CONTRIBUTIONS Jose J. G. Marin https://orcid. Concept and design: Marta Alonso-­ Peña, Ricardo org/0000-0003-1186-6849 Espinosa-­Escudero, Elisa Herraez, Oscar Briz, Maria J. Monte, Jose J. G. Marin. Clinical samples and data: REFERENCES Maria Luisa Cagigal, Jesus M. Gonzalez-­Santiago, Aida 1. Al-­Busafi SA, Hilzenrat N. Mild hypertransaminasemia in pri- Ortega-­ Alonso, Conrado Fernandez Rodriguez, Luis mary care. ISRN Hepatol. 2013;2013:256426. 2. Vajro P, Maddaluno S, Veropalumbo C. 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S U P P O R T I N G I N F O R M AT I O N 2022;76:1259–­1274. https://doi.org/10.1002/ Additional supporting information can be found online hep.32517 in the Supporting Information section at the end of this article.