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

Hepatology. 2022;76:1259–1274. | 1259 wileyonlinelibrary.com/journal/hep ORIGINAL ARTICLE Beneficial effect of ursodeoxycholic acid in patients with acylCoA oxidase 2 (ACOX2) deficiency– associated hypertransaminasemia Marta AlonsoPeña1,2 | Ricardo EspinosaEscudero1 | Elisa Herraez1,3 | Oscar Briz1,3 | Maria Luisa Cagigal4 | Jesus M. GonzalezSantiago5 | Aida OrtegaAlonso6 | Conrado FernandezRodriguez7 | Luis Bujanda3,8 | Marta Calvo Sanchez9 | Delia D´Avola10 | MariaCarlota Londoño3,11,12 | Moises Diago13 | Jose C. FernandezCheca3,12,14,15 | Carmen GarciaRuiz3,12,14,15 | Raul J. Andrade3,6 | Frank Lammert16,17 | Jesus Prieto3,10 | Javier Crespo2 | Javier Juamperez18 | Alvaro DiazGonzalez2 | Maria J. Monte1,3 | Jose J. G. Marin1,3 1Experimental Hepatology and Drug Targeting, Institute for Biomedical Research, University of Salamanca, Salamanca, Spain 2Gastroenterology and Hepatology Department, Clinical and Translational Research in Digestive Diseases, Valdecilla Research Institute (IDIVAL), Marqués de Valdecilla University Hospital, Santander, Spain 3Center for the Study of Liver and Gastrointestinal Diseases (CIBEREHD), Carlos III National Institute of Health, Madrid, Spain 4Pathological Anatomy Service, Hospital Universitario Marqués de Valdecilla, Santander, Spain 5Department of Gastroenterology and Hepatology, University Hospital of Salamanca, Institute for Biomedical Research, Salamanca, Spain 6Liver Unit, Gastroenterology Service, Institute of Biomedical Research of Málaga, School of Medicine, University Hospital Virgen de la Victoria, Málaga, Spain 7Gastroenterology Unit, Fundación Hospital Alcorcón, Rey Juan Carlos University, Madrid, Spain 8Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, University of the Basque Country, San Sebastian, Spain 9Segovia General Hospital, Segovia, Spain 10Department of Medicine, Clinica Universidad de Navarra and Center for Applied Medical Research, University of Navarra, Pamplona, Spain 11Liver Unit, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spain 12Institute of Biomedical Research of Barcelona (IDIBAPS), Barcelona, Spain 13Valencia University General Hospital, Valencia, Spain 14Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain 15Research Center for Alcoholic Liver and Pancreatic Diseases (ALPD) and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, California, USA 16Department of Medicine II, Saarland University Medical Center, Homburg, Germany 17Health Sciences, Hannover Medical School, Hannover, Germany 18Pediatric Hepatology and Liver Transplantation Unit, Vall d’Hebron University Hospital, Universitat Autónoma de Barcelona, Barcelona, Spain Received: 15 November 2021 | Revised: 3 April 2022 | Accepted: 4 April 2022 DOI: 10.1002/hep.32517 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. Marta AlonsoPeña and Ricardo EspinosaEscudero share first authorship and have contributed equally. Maria J. Monte and Jose J.G. Marin are cosenior authors and have contributed equally. Abbreviations: ACOX, acylCoA oxidase; ADAH, ACOX2 deficiencyassociated 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; HPLCMS/MS, highperformance liquid chromatographymass spectrometry; MAF, minor allele frequency; NTCP, Na+- taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; ROS, reactive oxygen species; RTqPCR, reverse transcription followed by quantitative polymerase chain reaction; TCA, taurocholic acid; THCA, trihydroxycholestanoic acid; UDCA, ursodeoxycholic acid. 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1260 | RESPONSE OF ACOX2 DEFICIENCY TO UDCA INTRODUCTION Hypertransaminasemia is a common condition detected in primary care practice[1] whose most frequent etiologies in adults are nonalcoholic fatty liver disease, viral infections, consumption of toxic substances (mainly alcohol), autoimmune liver disease, celiac disease, iron overload, and Wilson disease.[2] Up to 15% of persistent hypertransaminasemia cases in adults are idiopathic.[3] Biopsy permits a diagnosis in most (87%) but not all cases.[4] Information on asymptomatic hypertransaminasemia in childhood is scarce, and identifying its cause is particularly difficult in pediatric patients due to the lack of accompanying clinical manifestations.[2] Thus, up to 13% of persistent hypertransaminasemia found in children remains cryptogenic.[5] AcylCoA oxidase 2 (ACOX2) is a peroxisomal enzyme involved in the shortening of the cholesterol sidechain during bile acid (BA) biosynthesis. Two cases of ACOX2 deficiency were reported in 2016.[6,7] The first case, reported at the International Liver CongressEASL (April 2016; Barcelona, Spain),[6] involved a 16yearold man with unexplained hypertransaminasemia (25fold upper normal limit) and no other associated Correspondence Jose J. G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno E.D. Lab231, 37007Salamanca, Spain. Email: [email protected] Funding information 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), cofunded 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” (OLDHEPAMARKER, 0348_CIE_6_E), Spain. We also acknowledge support from grants PID2019111669RBI100, PID2020115055RBI00 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” SGR20171112, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European DrugInduced Liver Injury Network. Marta AlonsoPeña was the recipient of a predoctoral fellowship from “Ministerio de Educación, Cultura y Deporte” (BOEA20159456; FPU14/00214) and a Mobility Grant for Short Stays from “Ministerio de Ciencia, Innovación y Universidades” (EST17/00186). Ricardo EspinosaEscudero 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 Abstract Background and Aims: A variant (p.Arg225Trp) of peroxisomal acylCoA oxidase 2 (ACOX2), involved in bile acid (BA) sidechain shortening, has been associated with unexplained persistent hypertransaminasemia and accumulation of C27BAs, mainly 3α,7α,12αtrihydroxy5βcholestanoic acid (THCA). We aimed to investigate the prevalence of ACOX2 deficiencyassociated hypertransaminasemia (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 hypertransaminasemia from 11 hospitals and 13 of their relatives, seven individuals with abnormally high C27BA levels (>50% of total BAs) were identified by highperformance liquid chromatographymass 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 HuH7 hepatoma cells with THCA, which was efficiently taken up, but not through BA transporters, increased reactive oxygen species production (flow cytometry), endoplasmic reticulum stress biomarkers (GRP78, CHOP, and XBP1S/XBP1U ratio), and BAXα expression (reverse transcription followed by quantitative polymerase chain reaction and immunoblot), whereas cell viability was decreased (tetrazolium saltbased cell viability test). THCAinduced cell toxicity was higher than that of major C24BAs and was not prevented by UDCA. Fourteen predicted ACOX2 variants were generated (sitedirected mutagenesis) and expressed in HuH7 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 identified by a noninvasive diagnostic strategy based on plasma BA profiling and ACOX2 sequencing. Moreover, UDCA treatment can efficiently attenuate liver damage in these patients. 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 1261 HEPATOLOGY symptoms. In this individual, a homozygous variant (NM_003500.4:c.673C>T) in ACOX2, resulting in the amino acid change p.Arg225Trp, caused a decrease in enzymatic activity, together with reduced plasma levels of C24BAs and the accumulation of C27BAs.[8] The minor allele frequency (MAF) for c.673C>T in the Genome Aggregation Database (gnomAD v3.1), including more than 76,000 whole genomes, is 0.03%, which suggested that this is not a frequent variant found in the general population. However, data from the Medical Genome Project obtained recently in a Spanish cohort of healthy individuals (approximately 2000 genomes) showed an MAF of 0.7% for the c.673C>T allele,[9] suggesting that ACOX2 deficiency may not be an extremely rare condition. Indeed, in July 2016, a second case was reported by Vilarinho et al.,[7] which was followed by a third case identified 2 years later by Ferdinandusse et al.[10] Both children were born from consanguineous parents and died during childhood suffering from severe hepatic and neurological alterations. One of these patients reported carried a homozygous variant (c.207T>A) encoding truncated ACOX2 at codon 69 [NM_003500.4:c.207T>A (p.Tyr69Ter)],[7] whereas the other was homozygous for a four nucleotide deletion (NM_003500.4:c.461_464del), leading to a premature stop codon (p.Thr154fs).[10] Although elevated serum C27BA levels were found, these patients did not present a significant reduction in C24BA levels. Both cases showed a markedly higher severity in comparison with that caused by c.673C>T.[8] BA accumulation can lead to severe hepatocellular damage, which is often accompanied by inflammatory processes.[11] We have previously shown that the most abundant C27BAs in the plasma of these patients were 3α,7α,12αtrihydroxy5βcholestanoic acid (THCA) and its conjugated derivatives, which induces oxidative stress and cell death in cells overexpressing the c.673C>T ACOX2 variant.[8] Moreover, some BAs are capable of inducing endoplasmic reticulum (ER) stress,[12] releasing Ca2+ from the ER of hepatocytes, triggering Ca2+- dependent apoptosis[12] and activating the generation of reactive oxygen species (ROS) by mitochondria,[13] resulting in cell death,[14] which plays a pivotal role in the hypercholanemiainduced hepatocellular damage observed in several liver diseases. To elucidate the prevalence of ACOX2 deficiencyassociated hypertransaminasemia (ADAH), we analyzed a group of 33 such patients from different hospitals. We have also explored the mechanisms underlying THCAinduced toxicity. Furthermore, because several variants that may affect ACOX2 function have been predicted,[15] we selected those with the highest likelihood of impairing ACOX2 function, whose ability to affect BA biosynthesis was then evaluated in vitro. Finally, and more importantly, we have evaluated the clinical response of patients with ADAH to ursodeoxycholic acid (UDCA) treatment. MATERIALS AND METHODS Reagents and cell lines BArelated compounds were from SigmaAldrich (Merck, Madrid, Spain), except THCA and 7αhydroxy4cholesten3one (C4), which were from Avanti Polar Lipids (Alabaster, AL). Cells used in this study were human embryonic kidney (HEK) 293T, HuH7 (human hepatocellular carcinoma), HepG2 and HuH6 (human hepatoblastoma), IHH (immortalized human hepatocytes),[16] and stably transfected Chinese hamster ovary (CHO),[17] whose origin and culture conditions, as well as the rest of detailed information regarding the materials and methods, including statistical analysis, is available in the Supporting Information. Human samples Plasma and white blood cells samples were collected between 2016 and 2022 in 10 hospitals in Spain and one in Germany (Figure S1). The research protocol conformed to ethical guidelines of the 1975 Declaration of Helsinki. The use of genetic information was approved by the Human Ethical Committees of the University of Salamanca, Marqués de Valdecilla University Hospital, and Vall d'Hebron University Hospital (Spain). Informed consent was obtained from patients and relatives entering this study. Analytical methods Plasma samples were collected after overnight fasting. BA[8,18,19] and C4[20,21] concentrations were measured by highperformance liquid chromatographymass spectrometry (HPLCMS/MS). Plasma activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gammaglutamyltranspeptidase and alkaline phosphatase, as well as total and direct bilirubin concentrations, were measured using standard clinical automatic analyzers. Genetic analysis and gene expression DNA was obtained from blood samples to amplify the coding sequence of ACOX2 by highfidelity polymerase chain reaction (PCR). The amplified fragments containing both the exons and the exonintron boundaries from at least two reactions of PCR per exon were purified by agarose gel electrophoresis. Then, the amplicons were sequenced in both directions using forward and reverse primers as previously described.[8] Total RNA extraction from cells, reverse transcription (RT), and quantitative PCR (qPCR) were performed 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1262 | RESPONSE OF ACOX2 DEFICIENCY TO UDCA as previously reported[22] using the appropriate primers (Table S1). The results of mRNA abundance of target genes in each sample were normalized using hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression. Histological assessment Liver biopsies were processed routinely.[23] They were fixed with 10% neutral formalin and embedded in paraffin. Serial sections (4 µm) or mirrorimage sections were stained with hematoxylin and eosin or Masson’s trichrome (Figure S2). Histopathological findings and the fibrosis stage were graded by an expert pathologist using Knodell’s and Scheuer’s scores. Immunohistochemistry, immunoblotting, and immunofluorescence Immunostaining of ACOX2 and ACOX3 in 4µm sections of paraffinembedded liver biopsies was performed using Protein Atlas[24] validated antibodies antiACOX2 (HPA064845, Merck, Madrid) and antiACOX3 (HPA035840, Merck, Madrid), whose specificity was tested elsewhere.[10] Immunoblotting analyses of cell lysates were carried out with primary antibodies diluted in phosphatebuffered salineTween using blocking agents as appropriate (Table S2). glyceraldehyde3phosphate dehydrogenase (GAPDH) expression was used as a loading control. For immunofluorescence studies, cells were fixed with paraformaldehyde and permeabilized with Triton X100. After blocking with fetal bovine serum, cells were incubated for 1 h with antiACOX2 antibody (PA550297, Invitrogen, Thermo Fisher) and anticatalase antibody (LFMA0004, Invitrogen, Thermo Fisher). Finally, samples were incubated with the appropriate secondary antibodies, mouse or rabbit antiIgG conjugated with Alexa Fluor488 or Alexa Fluor594 (Invitrogen, Thermo Fisher) and 4,6diamidino2phenylindole (Invitrogen, Thermo Fisher). Visualization of the labeling was performed under a confocal microscope (TCS SP5, Leica, Barcelona). Functional study of ACOX2 variants The ACOX2 open reading frame (ORF) was amplified from human liver RNA and cloned into the pGEMT Easy vector (Promega, Madrid), which was used to generate vectors containing different ACOX2 variants by sitedirected mutagenesis (Table S3). The mutant ORF was then transferred to lentiviral vectors containing a V5tag.[8] Recombinant lentiviruses were produced in host HEK293T cells[25] and viral titers were determined by analyzing Enhnaced Green Fluorescent Potein (EGFP)- positive cells in a FACSCalibur flow cytometer (BD Biosciences, Madrid).[8] HuH7 hepatoma target cells were transduced with lentiviral vectors. Overexpression of ACOX2 variants was assessed by RTqPCR and immunoblotting. Double limitingdilution was performed to obtain monoclonal populations, which were selected according to ACOX2 mRNA and protein expression, as determined by RTqPCR, immunoblot, immunofluorescence, and BA metabolism studies. ACOX2 activity was assessed by studying the conversion of THCA into cholic acid (CA) in HuH7 cells expressing each variant. Cells were incubated with 2 µM THCA for 72 h. Biotransformation of THCA into CA was measured by analyzing them in cells and culture medium.[8] Toxicity and cell stress studies Cell viability was determined by the tetrazolium saltbased cell viability test using thiazolyl blue tetrazolium bromide (SigmaAldrich, Merck). Oxidative stress was determined by flow cytometry using 2′,7′- dichlorofluorescein diacetate (SigmaAldrich, Merck). ER stress and apoptosis were evaluated by analyzing atypical XBP1 splicing by RTqPCR by combining specific primers (Table S1) for total (XBP1total), short (XBP1S) and long (XBP1U) mRNA. Upregulation of genes involved in ER stress (CHOP and GRP78) and apoptosis activation (BAX and BCL2) was determined by RTqPCR (Table S1) and/or immunoblot (Table S2) after incubation with assayed agents for 24 h. Thapsigargin (2 μM) and sorafenib (5 µM) were used as a positive controls of ER stress and apoptosis activation, respectively. In silico prediction of dysfunctional ACOX2 variants The gnomAD v3.1 (exomes) (https://gnomad.broad insti tute.org) was searched to identify ACOX2 variants described in the population that might result in a dysfunctional protein. Inclusion criteria for the study of ACOX2 genetic variants were (i) single nucleotide polymorphism; (ii) MAF <1%; and (iii) generation of a missense variant. The selected variants were classified according to the prediction of the effect of each variant in ACOX2 activity, using SIFT and PolyPhen2 algorithms. The predicted functional impact score was calculated as follows: SIFT and PolyPhen2 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 of which 0 indicated the variant was 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 1263 HEPATOLOGY most likely benign and 4 indicated a high probability of deleterious effects. Transport assays BA uptake by HuH7 cells and transportermediated uptake by CHO cells was determined as previously described.[26] HuH7 and CHO cells, either wildtype or stably expressing Na+- taurocholate cotransporting polypepetide (NTCP), organic anion transporting polypeptide1B1 (OATP1B1), or OATP1B3,[17,27– 29] were incubated with uptake medium containing 50 µM THCA, glycocholic acid (GCA), or CA with or without 250 µM taurocholic acid (TCA) for 60 min. Uptake was stopped by rinsing the wells twice with cold uptake medium and twice with cold PBS. Cells were lysed with ultrapure water containing 5 µM of taurochenodeoxycholic acid (TCDA; internal standard), and BA concentrations were determined by HPLCMS/MS as described above. RESULTS Patients with idiopathic hypertransaminasemia Thirtythree patients with persistent hypertransaminasemia of unknown origin entered this study after ruling out common causes of elevated serum aminotransferases (viral hepatitis, alcoholic or metabolic liver damage, autoimmune hepatitis, hemochromatosis, Wilson's disease, alpha 1 antitrypsin deficiency, and obstructive cholestasis). Fourteen of these subjects showed hypercholanemia (≥10 µM), suggesting some degree of cholestasis. Among the rest of normocholanemic individuals, a C27BAs/C24BAs ratio >1 was found in four subjects, who were considered as potential ADAH cases. Thirteen relatives from these patients were also analyzed. Among them, three individuals had enhanced C27BAs levels together with genetic data consistent with dysfunctional ACOX2 (Figure 1). Plasma BA profiles of the individuals with confirmed ADAH are shown in Table 1. No significant alteration was detected in some cases in which serum C4 levels were measured (Table 1). Case 1 A 17yearold boy was first seen in 2010 in the Hepatology Unit of Marqués de Valdecilla University Hospital (Santander, Spain) due to fatigue symptoms and unexplained hypertransaminasemia (Table S4). Abdominal ultrasound showed normal liver and biliary tract. A liver biopsy (2010) revealed the presence of a single portal fibrous enlargement with focal portoportal bridging fibrosis and minimal portal inflammatory component, which suggested a preliminary diagnosis of seronegative autoimmune hepatitis. Accordingly, the patient received immunosuppressive drugs for 10 years, but biochemical remission was not achieved. Lack of response and persistent hypertransaminasemia during this time prompted new clinical evaluation. Again, all common causes of liver and biliary tract disorder were ruled out. A new liver biopsy (2019) showed no additional information (Figure S2). Finally, as the first episode of hypertransaminasemia had been recorded after treating a testicular torsion with nonsteroidal antiinflammatory drugs, which resembled the first case of ADAH previously described,[6,8] following an empiric approach, UDCA therapy was started. After 3 weeks, plasma aminotransferases reverted to normal (Table S4), and fatigue symptoms improved. Further analysis revealed that the patient showed an increased proportion of C27BAs (Figure 1, Table 1) and the presence in homozygosis of the previously described[8] ACOX2 variant c.673C>T (p.Arg225Trp) (Figure 1). The study of his family revealed that his brother, mother, and uncle carried this variant in homozygosis and accumulated C27BAs in serum (Figure 1, Table 1). Interestingly, all had undergone a cholecystectomy because of complicated cholelithiasis. Biochemical analysis of the uncle revealed hypertransaminasemia (ALT/AST: 131/68 U/l), which led his physician to prescribe him UDCA therapy. This restored his aminotransferase levels to normal values after 3 weeks (ALT/AST: 27/19 U/l). Serum aminotransferase levels were still low after 6 months of treatment (ALT/AST: 17/20 U/l), even though the abnormal proportion of C27BAs was not corrected (Figure 1, Table 1). In contrast, the father, who was heterozygous for the same variant, and the aunt, who did not carry any ACOX2 variant, were negative for C27BA accumulation (Figure 1, Table S8). Case 2 A 17yearold boy with persistent and unexplained hypertransaminasemia was first seen in the Hepatology Unit of Marqués de Valdecilla University Hospital in 2020. He had previously been diagnosed with growth hormone deficiency, treated with somatropin, and followed up by his pediatrician in another center. Hypertransaminasemia had been recorded since his first year of life (Table S5). Usual and unusual causes of persisting hypertransaminasemia were discarded, including lysosomal acid lipase deficiency. Abdominal ultrasound and MRCP showed no relevant findings. Liver biopsy showed no remarkable signs: slight sinusoidal dilatation and minimal central vein fibrosis (Figure S2). The analysis of plasma BAs revealed an increased proportion of C27BAs (Figure 1, Table 1). The genetic studies showed that he was homozygous for the ACOX2 variant c.673C>T (p.Arg225Trp) (Figure 1). UDCA treatment was initiated, achieving normalization of aminotransferases (Table S5) and 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1264 | RESPONSE OF ACOX2 DEFICIENCY TO UDCA reduction in the proportion of C27BAs (Figure 1) after 3 weeks. Nevertheless, the followup for 6 months of treatment revealed abnormal abundance of serum C27BAs (Figure 1, Table 1), despite the correction of serum aminotransferase levels was maintained (Table S5). The analysis of his family showed that his younger brother was homozygous for wildtype ACOX2, whereas the parents were heterozygotic carriers for c.673C>T (p.Arg225Trp) (Figure 1). The plasma BA profiles in the family members analyzed were normal. C24BA plasma levels were within the normal range in the mother and brother, whereas the father presented hypercholanemia (22 µM total BAs) with general enhanced levels of all BA molecular species but without an elevated proportion of C27BAs versus C24BAs (Figure 1; Table S9). Case 3 A 15yearold boy in followup at Santa Creu i Sant Pau Hospital (Barcelona, Spain) since the first years of life due to abdominal distension, steatorrhea, and hypertransaminasemia was first seen in the Pediatric Hepatology and Liver Transplantation Unit from Vall d’Hebron Hospital (Barcelona, Spain) in 2017. All clinical studies performed were negative, including the analysis of classic inborn errors of BA biosynthesis. An abdominal ultrasound revealed discrete hepatic hyperechogenicity, and a liver biopsy showed slight fibrosis (Figure S2). UDCA was empirically administered before ADAH was diagnosed. The treatment resulted in the normalization of plasma aminotransferases (Table S6) and symptoms relief. Then, BA profiling (Table 1) and FIGURE 1 Genealogical tree of families with members showing acylCoA oxidase (ACOX) 2 deficiencyassociated hypertransaminasemia (ADAH) (left panels) and the proportion of plasma concentrations of endogenous bile acids (BAs) with short sidechain (C24BAs) and immature BAs with unshorten sidechain (C27BAs) (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 C24BA concentrations. 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 1265 HEPATOLOGY TABLE 1 Bile acid species in serum collected from patients with acylCoA oxidase (ACOX) 2 deficiency– associated hypertransaminasemia (ADAH) UDCA treatment (months) Controls Case 1 Uncle case 1 Case 2 Case 3 Case 4 None 5None 0.75 6None 0.75 3641 47 None 0.75 511 7αOH4cholesten3one (C4) (nM) 17 ± 3 16 12 11 38 916 611 ND ND ND ND ND ND Endogenous C24bile acids (nM) 2290 ± 500 980 610 1137 1895 620 13,545 295 1336 960 600 703 1454 656 649 Endogenous C27bile acids (nM) 20 ± 3 1304 1334 2201 6122 791 5581 153 5014 2315 795 3245 2092 307 512 C27bile 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 C24bile acid species (nM) Glycocholic 117 ± 40 43 66 152 615 18 607 13 227 163 30 102 134 27 15 Glycochenodeoxycholic 421 ± 90 256 201 662 1018 40 2289 84 676 461 129 284 664 460 67 Glycodeoxycholic 180 ± 44 27 274 31 11 450 19 144 5 8 101 71 233 Glycolithocholic 9 ± 2 4 <1 46 724 22 14 1 5 1 6 1 18 Glycoursodeoxycholic 51 ± 17 5287* 67998* 4232* 16 13,065* 1667* 6264* 9949* 2524* 107 9281* 3305* 1477* Taurocholic 18 ± 7 1 5 11 57 <1 24 <1 21 1 5 14 11 1 4 Taurochenodeoxycholic 53 ± 11 14 22 50 88 460 275 44 12 57 83 29 7 Taurodeoxycholic 23 ± 6 3 1 9 7 1 6 1 13 2 1 26 8 1 2 Taurolithocholic 1 ± 1 <1 <1 32<1 <1 <1 2<1 <1 <1 1<1 1 Tauroursodeoxycholic 2 ± 1 97* <1 117* 201* <1 189* 15* 230* 235* 63* 7263* 64* 33* Taurosulfolithocholic 103 ± 15 3 4 78 3818 10 72 31 205 14 11 1189 Cholic 403 ± 92 122 276 547 504 6546 63 27 93 56 633 18 153 Chenodeoxycholic 330 ± 180 445 14 20 11 62755 32 19 140 119 41 311 110 46 Deoxycholic 417 ± 124 59 413 57781 34 29 816 45 114 467 Lithocholic 93 ± 27 1 1 14 415 17 16 113 910 148 Ursodeoxycholic 65 ± 21 6737* 27645* 96* 3105,873* 1276* 223* 3197* 2725* 23 2812* 2555* 1296* C27bile acid species (nM) Glycotrihydroxycholestanoic <1 118 156 189 763 38 1133 24 521 290 59 153 167 27 54 Glycodihydroxycholestanoic <1 15 39 127 117 19 79 861 30 718 33 <1 3 Taurotrihydroxycholestanoic 16 ± 3 706 902 1348 4492 553 3199 71 3443 1575 597 2125 1432 194 291 Taurodihydroxycholestanoic <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. UDCAassociated 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. 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1266 | RESPONSE OF ACOX2 DEFICIENCY TO UDCA directed genetic studies were performed. The patient showed an increased proportion of plasma C27BAs (Figure 1) and two heterozygous variants, presumably on two different alleles of ACOX2, the c.673C>T variant and a deletion of 4 nucleotides (c.456_459del) (Figure 1). This was predicted to generate the same protein change (p.Thr154fs) that had been previously described for a similar variant identified in ACOX2 by Ferdinandusse et al.[10] Liver function tests and analysis of BA profile after another 6 months of UDCA treatment revealed maintained normalization of aminotransferases besides a persistently altered BA profile (Figure 1, Table 1, and Table S6). The analysis of the proband’s family showed that the younger brother was homozygous for wildtype ACOX2, whereas the father was heterozygous for c.456_459del (p.Thr154fs) and the mother showed the c.673C>T (p.Arg225Trp) variant in heterozygosis (Figure 1). Their plasma BA profiles were normal (Figure 1, Table S10), with C27BAs and C24BAs within normal ranges. Case 4 A 7yearold boy presented a history of congenital heart disease (mild mitral stenosis, and moderatesevere aortic insufficiency) with slight developmental delay and oscillating hypertransaminasemia (Table S7). He was referred to the Pediatric Hepatology and Liver Transplantation Unit from Vall d’Hebron Hospital, where common causes of liver damage were ruled out. Abdominal ultrasound showed slight hepatic hyperechogenicity. Liver biopsy presented subtle polygonal morphology of the hepatocytes with no other remarkable findings (Figure S2). Due to the multiorgan affectation, a complete exome sequencing study was indicated. This showed the same double heterozygous variant of ACOX2 as in Case 3; i.e., variants c.673C>T (p.Arg225Trp) and c.456_459del (p.Thr154fs) (Figure 1). Plasma BA profiling (Table 1) confirmed enhanced C27BA proportion (Figure 1). Diagnosis of ADAH prompted UDCA treatment, resulting in the normalization of aminotransferase levels (Table S7) and a partial reduction in the proportion of C27BAs in plasma (Figure 1, Table 1). The analysis of the proband’s family members showed that the father carried the c.456_459del (p.Thr154fs) variant in heterozygosis and the mother presented the c.673C>T (p.Arg225Trp) variant in heterozygosis (Figure 1). In both parents, plasma C27BA and C24BA levels were within normal ranges (Figure 1; Table S11). FIGURE 2 Immunohistochemistry analysis of acylCoA 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. 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 1267 HEPATOLOGY Immunohistochemistry studies Immunohistochemistry of liver biopsies showed a complete absence of ACOX2 staining in patients with ADAH (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 have been included in Figure 2 as controls. In contrast, immunohistochemistry for ACOX3, another acylCoA oxidase that does not metabolize C27BA,[10] showed positive signal (with different intensity) in hepatocytes in all cases (Figure S3). C27BA– induced toxicity To analyze the cytotoxic effects of THCA, the main unconjugated C27BA synthesized in ADAH cases, we first analyzed the ability of THCA to enter liver cells. CA and GCA uptake by HuH7 cells was inhibited by TCA, which was also taken up by these cells. THCA uptake was higher and not affected by TCA (Figure 3A). CA, GCA, and TCA uptake by CHO cells was markedly enhanced by NTCP expression (Figure 3B) but very moderately by OATP1B1 (Figure 3C) and OATP1B3 (Figure 3D) expression. In all circumstances, a substantial THCA uptake was found, which was only slightly increased by NTCP expression and was not inhibited by TCA; THCA ability to induce oxidative stress was tested in liver cells. THCAinduced ROS production was higher than that induced by C24BAs, such as CA, chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA) in HuH7 and HuH6 cells, whereas HepG2 and IHH cells showed resistance to BAinduced oxidative stress (Figures 4A– D). To study the effect on ER stress, cells sensitive (HuH7) and resistant (HepG2) to BAinduced oxidative stress were selected. THCA induced a strong effect, which in most tests was stronger than that caused by C24BAs (Figure 4E– K). The reduction in viability of HepG2 and HuH7 cells after incubation with THCA was concentrationdependent in both cell lines, with HepG2 being less sensitive than HuH7 to THCAinduced cell death (Figure 4L,M). To confirm the link between THCAtoxicity and the presence of the p.Arg225Trp variant in ACOX2, monoclonal HuH7 cell sublines overexpressing wildtype ACOX2 (ACOX2WT) or the p.Arg225Trp mutated variant (ACOX2V1) were generated (Figure 5). ACOX2WT overexpression partly protected cells from THCAinduced cytotoxicity, whereas this C27BA caused a similar reduction in cell viability in control (Mock) and ACOX2V1 cells (Figure 4N). THCAinduced oxidative stress was not prevented by coincubation with UDCA (Figure 4O). Moreover, THCA markedly upregulated BAX mRNA, which was not reduced by UDCA (Figure 4P), whereas BCL2 was scarcely expressed by HuH7 cells (Figure 4Q). Consistently, THCAinduced cell death was not inhibited by UDCA (Figure 4R). Potentially harmful ACOX2 variants The search for variants that could cause ADAH, carried out in the databases, led to the selection of 46 variants, of which most (32 variants) had an extremely 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 the study and attention was focused on the functional analysis of the remaining 14 variants (Table 2). This specific set included the c.673C>T (p.Arg225Trp) variant initially identified (ACOX2V1). According to SIFT and PolyPhen2 predictions, only 11 of these variants are expected to have a functional impact on ACOX2 enzymatic activity. These predictions were further validated by measuring the ability of the variants, when expressed in HuH7 cells (Figure 5A,B), to enhance THCA biotransformation into CA. Monoclonal HuH7 cells stably overexpressing ACOX2WT or ACOX2V1 were used as positive and negative controls, respectively (Figure 5C,D). The activity of variants V11 and V14 was similar to that of ACOX2WT, suggesting that these variants do not have a functional impact on enzyme activity, as was predicted by in silico analysis. In contrast, variants V5 and V8 showed a moderately decreased activity, whereas variants V2, V4, V6, and V10 were significantly less functional (Figure 5E). DISCUSSION This study identified seven individuals from four unrelated families carrying ACOX2 variants involved in ADAH. Among them, five individuals showed persistent oscillating hypertransaminasemia. Three patients were homozygous carriers of c.673C>T (p.Arg225Trp), the same variant found in the first ADAH case reported,[8] whereas the other two carried heterozygous c.673C>T and c.456_459del ACOX2 variants in different alleles. Interestingly, the latter generates p.Thr154fs, which results in a premature stop codon and a truncated protein. This change is also caused by another similar deletion (c.461_464del) in ACOX2 mRNA described.[10] In contrast to our patients, the subject of that study carried the deletion in homozygosity and presented a more severe condition, with neurological symptoms and affection of lung, liver, heart, and muscle functions, and only survived 6 months.[10] The presence of other congenital alterations responsible for the multiorgan dysfunction of this case was not ruled out. The clinical conditions of the six patients with ADAH we have described so far 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1274 | RESPONSE OF ACOX2 DEFICIENCY TO UDCA 38. Vasavan T, Ferraro E, Ibrahim E, Dixon P, Gorelik J, Williamson C. Heart and bile acids - clinical consequences of altered bile acid metabolism. Biochim Biophys Acta Mol Basis Dis. 2018;1864(4):1345– 55. 39. Ferdinandusse S, Denis S, Dacremont G, Wanders RJ. Toxicity of peroxisomal C27bile acid intermediates. Mol Genet Metab. 2009;96(3):121– 8. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: AlonsoPeña M, EspinosaEscudero R, Herraez E, Briz O, Cagigal ML, GonzalezSantiago JM, et al. Beneficial effect of ursodeoxycholic acid in patients with acylCoA oxidase 2 (ACOX2) deficiency– associated hypertransaminasemia. Hepatology. 2022;76:1259– 1274. https://doi.org/10.1002/ hep.32517 15273350, 2022, 5, Downloaded from https://aasldpubs.onlinelibrary.wiley.com/doi/10.1002/hep.32517 by Universidad Del Pais Vasco, Wiley Online Library on [09/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License