Hepa ology. 2022;76:1259–1274.
|
1259
wileyonlinelib a y.com/jou nal/hep
ORIGINAL ARTICLE
Bene icial e ec o u sodeoxycholic acid in pa ien s
wi h acyl- CoA oxidase 2 (ACOX2) de iciency– associa ed
hype ansaminasemia
Ma a Alonso- Peña1,2 | Rica do Espinosa- Escude o1 | Elisa He aez1,3 |
Osca B iz1,3 | Ma ia Luisa Cagigal4 | Jesus M. Gonzalez- San iago5 |
Aida O ega- Alonso6 | Con ado Fe nandez- Rod iguez7 | Luis Bujanda3,8 |
Ma a Cal o Sanchez9 | Delia D´A ola10 | Ma ia- Ca lo a Londoño3,11,12 |
Moises Diago13 | Jose C. Fe nandez- Checa3,12,14,15 | Ca men Ga cia- Ruiz3,12,14,15 |
Raul J. And ade3,6 | F ank Lamme 16,17 | Jesus P ie o3,10 | Ja ie C espo2 |
Ja ie Juampe ez18 | Al a o Diaz- Gonzalez2 | Ma ia J. Mon e1,3 |
Jose J. G. Ma in1,3
1Expe imen al Hepa ology and D ug Ta ge ing, Ins i u e o Biomedical Resea ch, Uni e si y o Salamanca, Salamanca, Spain
2Gas oen e ology and Hepa ology Depa men , Clinical and T ansla ional Resea ch in Diges i e Diseases, Valdecilla Resea ch Ins i u e (IDIVAL), Ma qués de
Valdecilla Uni e si y Hospi al, San ande , Spain
3Cen e o he S udy o Li e and Gas oin es inal Diseases (CIBEREHD), Ca los III Na ional Ins i u e o Heal h, Mad id, Spain
4Pa hological Ana omy Se ice, Hospi al Uni e si a io Ma qués de Valdecilla, San ande , Spain
5Depa men o Gas oen e ology and Hepa ology, Uni e si y Hospi al o Salamanca, Ins i u e o Biomedical Resea ch, Salamanca, Spain
6Li e Uni , Gas oen e ology Se ice, Ins i u e o Biomedical Resea ch o Málaga, School o Medicine, Uni e si y Hospi al Vi gen de la Vic o ia, Málaga, Spain
7Gas oen e ology Uni , Fundación Hospi al Alco cón, Rey Juan Ca los Uni e si y, Mad id, Spain
8Depa men o Li e and Gas oin es inal Diseases, Biodonos ia Heal h Resea ch Ins i u e, Donos ia Uni e si y Hospi al, Uni e si y o he Basque Coun y,
San Sebas ian, Spain
9Sego ia Gene al Hospi al, Sego ia, Spain
10Depa men o Medicine, Clinica Uni e sidad de Na a a and Cen e o Applied Medical Resea ch, Uni e si y o Na a a, Pamplona, Spain
11Li e Uni , Hospi al Clínic de Ba celona, Uni e si y o Ba celona, Ba celona, Spain
12Ins i u e o Biomedical Resea ch o Ba celona (IDIBAPS), Ba celona, Spain
13Valencia Uni e si y Gene al Hospi al, Valencia, Spain
14Consejo Supe io de In es igaciones Cien í icas (CSIC), Mad id, Spain
15Resea ch Cen e o Alcoholic Li e and Panc ea ic Diseases (ALPD) and Ci hosis, Keck School o Medicine, Uni e si y o Sou he n Cali o nia, Los
Angeles, Cali o nia, USA
16Depa men o Medicine II, Saa land Uni e si y Medical Cen e , Hombu g, Ge many
17Heal h Sciences, Hanno e Medical School, Hanno e , Ge many
18Pedia ic Hepa ology and Li e T ansplan a ion Uni , Vall d’Heb on Uni e si y Hospi al, Uni e si a Au ónoma de Ba celona, Ba celona, Spain
Recei ed: 15 No embe 2021
|
Re ised: 3 Ap il 2022
|
Accep ed: 4 Ap il 2022
DOI: 10.1002/hep.32517
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NonComme cial-NoDe i s License, which pe mi s use and dis ibu ion in
any medium, p o ided he o iginal wo k is p ope ly ci ed, he use is non-comme cial and no modi ica ions o adap a ions a e made.
© 2022 The Au ho s. Hepa ology published by Wiley Pe iodicals LLC on behal o Ame ican Associa ion o he S udy o Li e Diseases.
Ma a Alonso- Peña and Rica do Espinosa- Escude o sha e i s au ho ship and ha e con ibu ed equally.
Ma ia J. Mon e and Jose J.G. Ma in a e co- senio au ho s and ha e con ibu ed equally.
Abb e ia ions: ACOX, acyl- CoA oxidase; ADAH, ACOX2 de iciency- associa ed hype ansaminasemia; ALT, alanine amino ans e ase; AST, aspa a e
amino ans e ase; BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; CHO, Chinese hams e o a y; DCA, deoxycholic acid; ER, endoplasmic
e iculum; GCA, glycocholic acid; HPLC- MS/MS, high- pe o mance liquid ch oma og aphy- mass spec ome y; MAF, mino allele equency; NTCP, Na+-
au ochola e co anspo ing polypep ide; OATP, o ganic anion anspo ing polypep ide; ROS, eac i e oxygen species; RT- qPCR, e e se ansc ip ion
ollowed by quan i a i e polyme ase chain eac ion; TCA, au ocholic acid; THCA, ihyd oxycholes anoic acid; UDCA, u sodeoxycholic acid.
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1260
|
RESPONSE OF ACOX2 DEFICIENCY TO UDCA
INTRODUCTION
Hype ansaminasemia is a common condi ion de-
ec ed in p ima y ca e p ac ice[1] whose mos equen
e iologies in adul s a e nonalcoholic a y li e dis-
ease, i al in ec ions, consump ion o oxic subs ances
(mainly alcohol), au oimmune li e disease, celiac dis-
ease, i on o e load, and Wilson disease.[2] Up o 15%
o pe sis en hype ansaminasemia cases in adul s a e
idiopa hic.[3] Biopsy pe mi s a diagnosis in mos (87%)
bu no all cases.[4] In o ma ion on asymp oma ic hype -
ansaminasemia in childhood is sca ce, and iden i ying
i s cause is pa icula ly di icul in pedia ic pa ien s due
o he lack o accompanying clinical mani es a ions.[2]
Thus, up o 13% o pe sis en hype ansaminasemia
ound in child en emains c yp ogenic.[5]
Acyl- CoA oxidase 2 (ACOX2) is a pe oxisomal en-
zyme in ol ed in he sho ening o he choles e ol side-
chain du ing bile acid (BA) biosyn hesis. Two cases o
ACOX2 de iciency we e epo ed in 2016.[6,7] The i s
case, epo ed a he In e na ional Li e Cong ess-
EASL (Ap il 2016; Ba celona, Spain),[6] in ol ed a
16- yea - old man wi h unexplained hype ansaminase-
mia (2- 5- old uppe no mal limi ) and no o he associa ed
Co espondence
Jose J. G. Ma in, Depa men o
Physiology and Pha macology, Campus
Miguel de Unamuno E.D. Lab231,
37007- Salamanca, Spain.
Email: jjgma [email protected]
Funding in o ma ion
This s udy was suppo ed by he ollowing
g an s: CIBERehd (EHD15PI05/2016);
Fondo de In es igaciones Sani a ias,
Ins i u o de Salud Ca los III, Spain
(PI19/00819 and PI20/00189), co- unded
by Eu opean Regional De elopmen
Fund/Eu opean Social Fund, “In es ing
in you u u e”; “Jun a de Cas illa y León”
(SA074P20); Fundació Ma a o TV3
(201916– 31); AECC Scien i ic Founda ion
(2017/2020), Spain; and “Cen o
In e nacional sob e el En ejecimien o”
(OLD- HEPAMARKER, 0348_CIE_6_E),
Spain. We also acknowledge suppo
om g an s PID2019- 111669RBI- 100,
PID2020- 115055RB- I00 om Plan
Nacional de I+D unded by he “Agencia
Es a al de In es igación” (AEI) and he
cen e g an P50AA011999 Sou he n
Cali o nia Resea ch Cen e o ALPD
and Ci hosis unded by NIAAA/NIH,
as well as suppo om AGAUR o
he “Gene ali a de Ca alunya” SGR-
2017- 1112, Eu opean Coope a ion in
Science & Technology (COST) ACTION
CA17112 P ospec i e Eu opean D ug-
Induced Li e Inju y Ne wo k. Ma a
Alonso- Peña was he ecipien o a
p edoc o al ellowship om “Minis e io de
Educación, Cul u a y Depo e” (BOE- A-
2015- 9456; FPU- 14/00214) and a Mobili y
G an o Sho S ays om “Minis e io
de Ciencia, Inno ación y Uni e sidades”
(EST17/00186). Rica do Espinosa-
Escude o is he ecipien o a p edoc o al
ellowship om “Jun a de Cas illa y
León” and “Fondo Social Eu opeo”
(EDU/574/2018). The unding sou ces
we e no in ol ed in he esea ch design
o p epa a ion o he a icle
Abs ac
Backg ound and Aims: A a ian (p.A g225T p) o pe oxisomal acyl- CoA
oxidase 2 (ACOX2), in ol ed in bile acid (BA) side- chain sho ening, has been
associa ed wi h unexplained pe sis en hype ansaminasemia and accumu-
la ion o C27- BAs, mainly 3α,7α,12α- ihyd oxy- 5β- choles anoic acid (THCA).
We aimed o in es iga e he p e alence o ACOX2 de iciency- associa ed hy-
pe ansaminasemia (ADAH), i s esponse o u sodeoxycholic acid (UDCA),
elucida e i s pa hophysiological mechanism and iden i y o he inbo n e o s
ha could cause his al e a ion.
Me hods and Resul s: Among 33 pa ien s wi h unexplained hype ansami-
nasemia om 11 hospi als and 13 o hei ela i es, se en indi iduals wi h
abno mally high C27- BA le els (>50% o o al BAs) we e iden i ied by high-
pe o mance liquid ch oma og aphy- mass spec ome y. The p.A g225T p
a ian was ound in homozygosi y (exon ampli ica ion/sequencing) in wo
pa ien s and h ee amily membe s. Two addi ional non ela ed pa ien s
we e he e ozygous ca ie s o di e en alleles: c.673C>T (p.A g225T p) and
c.456_459del (p.Th 154 s). In pa ien s wi h ADAH, impai ed li e exp ession o
ACOX2, bu no ACOX3, was ound (immunohis ochemis y). T ea men wi h
UDCA no malized amino ans e ase le els. Incuba ion o HuH- 7 hepa oma
cells wi h THCA, which was e icien ly aken up, bu no h ough BA anspo -
e s, inc eased eac i e oxygen species p oduc ion ( low cy ome y), endo-
plasmic e iculum s ess bioma ke s (GRP78, CHOP, and XBP1- S/XBP1- U
a io), and BAXα exp ession ( e e se ansc ip ion ollowed by quan i a i e
polyme ase chain eac ion and immunoblo ), whe eas cell iabili y was de-
c eased ( e azolium sal - based cell iabili y es ). THCA- induced cell oxici y
was highe han ha o majo C24- BAs and was no p e en ed by UDCA.
Fou een p edic ed ACOX2 a ian s we e gene a ed (si e- di ec ed mu agen-
esis) and exp essed in HuH- 7 cells. Func ional es s o de e mine hei abili y
o me abolize THCA iden i ied six wi h he po en ial o cause ADAH.
Conclusions: Dys unc ional ACOX2 has been ound in se e al pa ien s wi h
unexplained hype ansaminasemia. This condi ion can be accu a ely iden i-
ied by a nonin asi e diagnos ic s a egy based on plasma BA p o iling and
ACOX2 sequencing. Mo eo e , UDCA ea men can e icien ly a enua e
li e damage in hese pa ien s.
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
|
1261
HEPATOLOGY
symp oms. In his indi idual, a homozygous a ian
(NM_003500.4:c.673C>T) in ACOX2, esul ing in he
amino acid change p.A g225T p, caused a dec ease
in enzyma ic ac i i y, oge he wi h educed plasma
le els o C24- BAs and he accumula ion o C27- BAs.[8]
The mino allele equency (MAF) o c.673C>T in he
Genome Agg ega ion Da abase (gnomAD 3.1), includ-
ing mo e han 76,000 whole genomes, is 0.03%, which
sugges ed ha his is no a equen a ian ound in
he gene al popula ion. Howe e , da a om he Medical
Genome P ojec ob ained ecen ly in a Spanish coho
o heal hy indi iduals (app oxima ely 2000 genomes)
showed an MAF o 0.7% o he c.673C>T allele,[9] sug-
ges ing ha ACOX2 de iciency may no be an ex emely
a e condi ion. Indeed, in July 2016, a second case was
epo ed by Vila inho e al.,[7] which was ollowed by
a hi d case iden i ied 2 yea s la e by Fe dinandusse
e al.[10] Bo h child en we e bo n om consanguine-
ous pa en s and died du ing childhood su e ing om
se e e hepa ic and neu ological al e a ions. One o
hese pa ien s epo ed ca ied a homozygous a ian
(c.207T>A) encoding unca ed ACOX2 a codon 69
[NM_003500.4:c.207T>A (p.Ty 69Te )],[7] whe eas he
o he was homozygous o a ou nucleo ide dele ion
(NM_003500.4:c.461_464del), leading o a p ema u e
s op codon (p.Th 154 s).[10] Al hough ele a ed se um
C27- BA le els we e ound, hese pa ien s did no p es-
en a signi ican educ ion in C24- BA le els. Bo h cases
showed a ma kedly highe se e i y in compa ison wi h
ha caused by c.673C>T.[8]
BA accumula ion can lead o se e e hepa ocellula
damage, which is o en accompanied by in lamma-
o y p ocesses.[11] We ha e p e iously shown ha he
mos abundan C27- BAs in he plasma o hese pa-
ien s we e 3α,7α,12α- ihyd oxy- 5β- choles anoic acid
(THCA) and i s conjuga ed de i a i es, which induces
oxida i e s ess and cell dea h in cells o e exp essing
he c.673C>T ACOX2 a ian .[8] Mo eo e , some BAs
a e capable o inducing endoplasmic e iculum (ER)
s ess,[12] eleasing Ca2+ om he ER o hepa ocy es,
igge ing Ca2+- dependen apop osis[12] and ac i a ing
he gene a ion o eac i e oxygen species (ROS) by mi-
ochond ia,[13] esul ing in cell dea h,[14] which plays a
pi o al ole in he hype cholanemia- induced hepa ocel-
lula damage obse ed in se e al li e diseases.
To elucida e he p e alence o ACOX2 de iciency-
associa ed hype ansaminasemia (ADAH), we an-
alyzed a g oup o 33 such pa ien s om di e en
hospi als. We ha e also explo ed he mechanisms un-
de lying THCA- induced oxici y. Fu he mo e, because
se e al a ian s ha may a ec ACOX2 unc ion ha e
been p edic ed,[15] we selec ed hose wi h he highes
likelihood o impai ing ACOX2 unc ion, whose abili y
o a ec BA biosyn hesis was hen e alua ed in i o.
Finally, and mo e impo an ly, we ha e e alua ed he
clinical esponse o pa ien s wi h ADAH o u sodeoxy-
cholic acid (UDCA) ea men .
MATERIALS AND METHODS
Reagen s and cell lines
BA- ela ed compounds we e om Sigma- Ald ich
(Me ck, Mad id, Spain), excep THCA and 7α- hyd oxy-
4- choles en- 3- one (C4), which we e om A an i Pola
Lipids (Alabas e , AL). Cells used in his s udy we e
human emb yonic kidney (HEK) 293T, HuH- 7 (human
hepa ocellula ca cinoma), HepG2 and HuH- 6 (human
hepa oblas oma), IHH (immo alized human hepa o-
cy es),[16] and s ably ans ec ed Chinese hams e
o a y (CHO),[17] whose o igin and cul u e condi ions,
as well as he es o de ailed in o ma ion ega ding he
ma e ials and me hods, including s a is ical analysis, is
a ailable in he Suppo ing In o ma ion.
Human samples
Plasma and whi e blood cells samples we e collec ed
be ween 2016 and 2022 in 10 hospi als in Spain and
one in Ge many (Figu e S1). The esea ch p o ocol con-
o med o e hical guidelines o he 1975 Decla a ion o
Helsinki. The use o gene ic in o ma ion was app o ed
by he Human E hical Commi ees o he Uni e si y o
Salamanca, Ma qués de Valdecilla Uni e si y Hospi al,
and Vall d'Heb on Uni e si y Hospi al (Spain). In o med
consen was ob ained om pa ien s and ela i es en e -
ing his s udy.
Analy ical me hods
Plasma samples we e collec ed a e o e nigh as ing.
BA[8,18,19] and C4[20,21] concen a ions we e measu ed
by high- pe o mance liquid ch oma og aphy- mass
spec ome y (HPLC- MS/MS). Plasma ac i i ies o ala-
nine amino ans e ase (ALT), aspa a e amino ans-
e ase (AST), gamma- glu amyl anspep idase and
alkaline phospha ase, as well as o al and di ec bili-
ubin concen a ions, we e measu ed using s anda d
clinical au oma ic analyze s.
Gene ic analysis and gene exp ession
DNA was ob ained om blood samples o ampli y he
coding sequence o ACOX2 by high- ideli y polyme ase
chain eac ion (PCR). The ampli ied agmen s con ain-
ing bo h he exons and he exon- in on bounda ies om
a leas wo eac ions o PCR pe exon we e pu i ied
by aga ose gel elec opho esis. Then, he amplicons
we e sequenced in bo h di ec ions using o wa d and
e e se p ime s as p e iously desc ibed.[8]
To al RNA ex ac ion om cells, e e se ansc ip ion
(RT), and quan i a i e PCR (qPCR) we e pe o med
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1262
|
RESPONSE OF ACOX2 DEFICIENCY TO UDCA
as p e iously epo ed[22] using he app op ia e p im-
e s (Table S1). The esul s o mRNA abundance o
a ge genes in each sample we e no malized using
hypoxan hine phospho ibosyl ans e ase 1 (HPRT1)
exp ession.
His ological assessmen
Li e biopsies we e p ocessed ou inely.[23] They we e
ixed wi h 10% neu al o malin and embedded in pa -
a in. Se ial sec ions (4 µm) o mi o - image sec ions
we e s ained wi h hema oxylin and eosin o Masson’s
ich ome (Figu e S2). His opa hological indings and
he ib osis s age we e g aded by an expe pa hologis
using Knodell’s and Scheue ’s sco es.
Immunohis ochemis y, immunoblo ing,
and immuno luo escence
Immunos aining o ACOX2 and ACOX3 in 4- µm sec ions
o pa a in- embedded li e biopsies was pe o med
using P o ein A las[24] alida ed an ibodies an i-
ACOX2 (HPA064845, Me ck, Mad id) and an i- ACOX3
(HPA035840, Me ck, Mad id), whose speci ici y was
es ed elsewhe e.[10] Immunoblo ing analyses o cell
lysa es we e ca ied ou wi h p ima y an ibodies dilu ed
in phospha e- bu e ed saline- Tween using blocking
agen s as app op ia e (Table S2). glyce aldehyde- 3-
phospha e dehyd ogenase (GAPDH) exp ession was
used as a loading con ol.
Fo immuno luo escence s udies, cells we e
ixed wi h pa a o maldehyde and pe meabilized wi h
T i on X- 100. A e blocking wi h e al bo ine se um,
cells we e incuba ed o 1 h wi h an i- ACOX2 an i-
body (PA5- 50297, In i ogen, The mo Fishe ) and
an ica alase an ibody (LF- MA0004, In i ogen,
The mo Fishe ). Finally, samples we e incuba ed
wi h he app op ia e seconda y an ibodies, mouse
o abbi an i- IgG conjuga ed wi h Alexa Fluo - 488
o Alexa Fluo - 594 (In i ogen, The mo Fishe ) and
4,6- diamidino- 2- phenylindole (In i ogen, The mo
Fishe ). Visualiza ion o he labeling was pe o med
unde a con ocal mic oscope (TCS SP5, Leica,
Ba celona).
Func ional s udy o ACOX2 a ian s
The ACOX2 open eading ame (ORF) was ampli ied
om human li e RNA and cloned in o he pGEM- T
Easy ec o (P omega, Mad id), which was used o
gene a e ec o s con aining di e en ACOX2 a i-
an s by si e- di ec ed mu agenesis (Table S3). The
mu an ORF was hen ans e ed o len i i al ec o s
con aining a V5- ag.[8] Recombinan len i i uses we e
p oduced in hos HEK293T cells[25] and i al i e s we e
de e mined by analyzing Enhnaced G een Fluo escen
Po ein (EGFP)- posi i e cells in a FACSCalibu low cy-
ome e (BD Biosciences, Mad id).[8] HuH- 7 hepa oma
a ge cells we e ansduced wi h len i i al ec o s.
O e exp ession o ACOX2 a ian s was assessed by
RT- qPCR and immunoblo ing. Double limi ing- dilu ion
was pe o med o ob ain monoclonal popula ions,
which we e selec ed acco ding o ACOX2 mRNA and
p o ein exp ession, as de e mined by RT- qPCR, im-
munoblo , immuno luo escence, and BA me abolism
s udies. ACOX2 ac i i y was assessed by s udying
he con e sion o THCA in o cholic acid (CA) in HuH- 7
cells exp essing each a ian . Cells we e incuba ed
wi h 2 µM THCA o 72 h. Bio ans o ma ion o THCA
in o CA was measu ed by analyzing hem in cells and
cul u e medium.[8]
Toxici y and cell s ess s udies
Cell iabili y was de e mined by he e azolium sal - based
cell iabili y es using hiazolyl blue e azolium b omide
(Sigma- Ald ich, Me ck). Oxida i e s ess was de e -
mined by low cy ome y using 2′,7′- dichlo o luo escein
diace a e (Sigma- Ald ich, Me ck). ER s ess and apop-
osis we e e alua ed by analyzing a ypical XBP1 splic-
ing by RT- qPCR by combining speci ic p ime s (Table
S1) o o al (XBP1- o al), sho (XBP1- S) and long
(XBP1- U) mRNA. Up- egula ion o genes in ol ed in
ER s ess (CHOP and GRP78) and apop osis ac i a-
ion (BAX and BCL2) was de e mined by RT- qPCR
(Table S1) and/o immunoblo (Table S2) a e incuba-
ion wi h assayed agen s o 24 h. Thapsiga gin (2 μM)
and so a enib (5 µM) we e used as a posi i e con ols o
ER s ess and apop osis ac i a ion, espec i ely.
In silico p edic ion o dys unc ional
ACOX2 a ian s
The gnomAD 3.1 (exomes) (h ps://gnomad.b oad
ins i u e.o g) was sea ched o iden i y ACOX2 a i-
an s desc ibed in he popula ion ha migh esul in a
dys unc ional p o ein. Inclusion c i e ia o he s udy o
ACOX2 gene ic a ian s we e (i) single nucleo ide pol-
ymo phism; (ii) MAF <1%; and (iii) gene a ion o a mis-
sense a ian . The selec ed a ian s we e classi ied
acco ding o he p edic ion o he e ec o each a ian
in ACOX2 ac i i y, using SIFT and PolyPhen- 2 algo-
i hms. The p edic ed unc ional impac sco e was cal-
cula ed as ollows: SIFT and PolyPhen- 2 p edic ions o
he e ec o each a ian in ACOX2 ac i i y was gi en
alues om 0 o 2, depending on hei p edic ed e -
ec by each algo i hm (null = 0, p obable = 1, o highly
p obable = 2). The sum o bo h sco es esul ed in a
scale om 0 o 4 o which 0 indica ed he a ian was
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
|
1263
HEPATOLOGY
mos likely benign and 4 indica ed a high p obabili y o
dele e ious e ec s.
T anspo assays
BA up ake by HuH- 7 cells and anspo e - media ed
up ake by CHO cells was de e mined as p e iously
desc ibed.[26] HuH- 7 and CHO cells, ei he wild- ype
o s ably exp essing Na+- au ochola e co anspo ing
polypepe ide (NTCP), o ganic anion anspo ing poly-
pep ide- 1B1 (OATP1B1), o OATP1B3,[17,27– 29] we e in-
cuba ed wi h up ake medium con aining 50 µM THCA,
glycocholic acid (GCA), o CA wi h o wi hou 250 µM
au ocholic acid (TCA) o 60 min. Up ake was s opped
by insing he wells wice wi h cold up ake medium and
wice wi h cold PBS. Cells we e lysed wi h ul a- pu e
wa e con aining 5 µM o au ochenodeoxycholic acid
(TCDA; in e nal s anda d), and BA concen a ions we e
de e mined by HPLC- MS/MS as desc ibed abo e.
RESULTS
Pa ien s wi h idiopa hic
hype ansaminasemia
Thi y- h ee pa ien s wi h pe sis en hype ansamina-
semia o unknown o igin en e ed his s udy a e uling
ou common causes o ele a ed se um amino ans-
e ases ( i al hepa i is, alcoholic o me abolic li e dam-
age, au oimmune hepa i is, hemoch oma osis, Wilson's
disease, alpha 1 an i ypsin de iciency, and obs uc i e
choles asis). Fou een o hese subjec s showed hype -
cholanemia (≥10 µM), sugges ing some deg ee o chol-
es asis. Among he es o no mocholanemic indi iduals,
a C27- BAs/C24- BAs a io >1 was ound in ou subjec s,
who we e conside ed as po en ial ADAH cases. Thi een
ela i es om hese pa ien s we e also analyzed. Among
hem, h ee indi iduals had enhanced C27- BAs le els
oge he wi h gene ic da a consis en wi h dys unc ional
ACOX2 (Figu e 1). Plasma BA p o iles o he indi idu-
als wi h con i med ADAH a e shown in Table 1. No sig-
ni ican al e a ion was de ec ed in some cases in which
se um C4 le els we e measu ed (Table 1).
Case 1
A 17- yea - old boy was i s seen in 2010 in he Hepa ology
Uni o Ma qués de Valdecilla Uni e si y Hospi al
(San ande , Spain) due o a igue symp oms and unex-
plained hype ansaminasemia (Table S4). Abdominal
ul asound showed no mal li e and bilia y ac . A li e
biopsy (2010) e ealed he p esence o a single po al
ib ous enla gemen wi h ocal po o- po al b idging ib o-
sis and minimal po al in lamma o y componen , which
sugges ed a p elimina y diagnosis o se onega i e au o-
immune hepa i is. Acco dingly, he pa ien ecei ed im-
munosupp essi e d ugs o 10 yea s, bu biochemical
emission was no achie ed. Lack o esponse and pe -
sis en hype ansaminasemia du ing his ime p omp ed
new clinical e alua ion. Again, all common causes o
li e and bilia y ac diso de we e uled ou . A new li e
biopsy (2019) showed no addi ional in o ma ion (Figu e
S2). Finally, as he i s episode o hype ansaminasemia
had been eco ded a e ea ing a es icula o sion wi h
nons e oidal an i- in lamma o y d ugs, which esembled
he i s case o ADAH p e iously desc ibed,[6,8] ollowing
an empi ic app oach, UDCA he apy was s a ed. A e
3 weeks, plasma amino ans e ases e e ed o no mal
(Table S4), and a igue symp oms imp o ed. Fu he anal-
ysis e ealed ha he pa ien showed an inc eased p o-
po ion o C27- BAs (Figu e 1, Table 1) and he p esence
in homozygosis o he p e iously desc ibed[8] ACOX2
a ian c.673C>T (p.A g225T p) (Figu e 1). The s udy o
his amily e ealed ha his b o he , mo he , and uncle ca -
ied his a ian in homozygosis and accumula ed C27-
BAs in se um (Figu e 1, Table 1). In e es ingly, all had
unde gone a cholecys ec omy because o complica ed
choleli hiasis. Biochemical analysis o he uncle e ealed
hype ansaminasemia (ALT/AST: 131/68 U/l), which led
his physician o p esc ibe him UDCA he apy. This e-
s o ed his amino ans e ase le els o no mal alues a e
3 weeks (ALT/AST: 27/19 U/l). Se um amino ans e ase
le els we e s ill low a e 6 mon hs o ea men (ALT/AST:
17/20 U/l), e en hough he abno mal p opo ion o C27-
BAs was no co ec ed (Figu e 1, Table 1). In con as ,
he a he , who was he e ozygous o he same a ian ,
and he aun , who did no ca y any ACOX2 a ian , we e
nega i e o C27- BA accumula ion (Figu e 1, Table S8).
Case 2
A 17- yea - old boy wi h pe sis en and unexplained hy-
pe ansaminasemia was i s seen in he Hepa ology
Uni o Ma qués de Valdecilla Uni e si y Hospi al in
2020. He had p e iously been diagnosed wi h g ow h
ho mone de iciency, ea ed wi h soma opin, and
ollowed up by his pedia ician in ano he cen e .
Hype ansaminasemia had been eco ded since his
i s yea o li e (Table S5). Usual and unusual causes
o pe sis ing hype ansaminasemia we e disca ded,
including lysosomal acid lipase de iciency. Abdominal
ul asound and MRCP showed no ele an indings.
Li e biopsy showed no ema kable signs: sligh si-
nusoidal dila a ion and minimal cen al ein ib osis
(Figu e S2). The analysis o plasma BAs e ealed an
inc eased p opo ion o C27- BAs (Figu e 1, Table 1).
The gene ic s udies showed ha he was homozy-
gous o he ACOX2 a ian c.673C>T (p.A g225T p)
(Figu e 1). UDCA ea men was ini ia ed, achie ing
no maliza ion o amino ans e ases (Table S5) and
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1264
|
RESPONSE OF ACOX2 DEFICIENCY TO UDCA
educ ion in he p opo ion o C27- BAs (Figu e 1) a e
3 weeks. Ne e heless, he ollow- up o 6 mon hs o
ea men e ealed abno mal abundance o se um
C27- BAs (Figu e 1, Table 1), despi e he co ec ion o
se um amino ans e ase le els was main ained (Table
S5). The analysis o his amily showed ha his younge
b o he was homozygous o wild- ype ACOX2,
whe eas he pa en s we e he e ozygo ic ca ie s o
c.673C>T (p.A g225T p) (Figu e 1). The plasma BA
p o iles in he amily membe s analyzed we e no mal.
C24- BA plasma le els we e wi hin he no mal ange in
he mo he and b o he , whe eas he a he p esen ed
hype cholanemia (22 µM o al BAs) wi h gene al en-
hanced le els o all BA molecula species bu wi hou
an ele a ed p opo ion o C27- BAs e sus C24- BAs
(Figu e 1; Table S9).
Case 3
A 15- yea - old boy in ollow- up a San a C eu i San
Pau Hospi al (Ba celona, Spain) since he i s yea s
o li e due o abdominal dis ension, s ea o hea, and
hype ansaminasemia was i s seen in he Pedia ic
Hepa ology and Li e T ansplan a ion Uni om Vall
d’Heb on Hospi al (Ba celona, Spain) in 2017. All clini-
cal s udies pe o med we e nega i e, including he
analysis o classic inbo n e o s o BA biosyn hesis. An
abdominal ul asound e ealed disc e e hepa ic hype -
echogenici y, and a li e biopsy showed sligh ib osis
(Figu e S2). UDCA was empi ically adminis e ed be o e
ADAH was diagnosed. The ea men esul ed in he
no maliza ion o plasma amino ans e ases (Table S6)
and symp oms elie . Then, BA p o iling (Table 1) and
FIGURE 1 Genealogical ee o amilies wi h membe s showing acyl- CoA oxidase (ACOX) 2 de iciency- associa ed
hype ansaminasemia (ADAH) (le panels) and he p opo ion o plasma concen a ions o endogenous bile acids (BAs) wi h sho side-
chain (C24- BAs) and imma u e BAs wi h unsho en side- chain (C27- BAs) ( igh panels). In indi iduals ea ed wi h u sodeoxycholic acid
(UDCA; 12 o 15 mg/kg/day), his BA and i s conjuga es we e no conside ed o calcula ing plasma endogenous C24- BA concen a ions.
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
|
1265
HEPATOLOGY
TABLE 1 Bile acid species in se um collec ed om pa ien s wi h acyl- CoA oxidase (ACOX) 2 de iciency– associa ed hype ansaminasemia (ADAH)
UDCA ea men (mon hs)
Con ols Case 1 Uncle case 1 Case 2 Case 3 Case 4
None 5None 0.75 6None 0.75 3641 47 None 0.75 511
7α- OH- 4- choles en- 3- one (C4) (nM) 17 ± 3 16 12 11 38 916 611 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 27 274 31 11 450 19 144 5 8 101 71 233
Glyco- li hocholic 9 ± 2 4 <1 46 724 22 14 1 5 1 6 1 18
Glyco- u sodeoxycholic 51 ± 17 5287* 67998* 4232* 16 13,065* 1667* 6264* 9949* 2524* 107 9281* 3305* 1477*
Tau o- cholic 18 ± 7 1 5 11 57 <1 24 <1 21 1 5 14 11 1 4
Tau o- chenodeoxycholic 53 ± 11 14 22 50 88 460 275 44 12 57 83 29 7
Tau o- deoxycholic 23 ± 6 3 1 9 7 1 6 1 13 2 1 26 8 1 2
Tau o- li hocholic 1 ± 1 <1 <1 32<1 <1 <1 2<1 <1 <1 1<1 1
Tau o- u sodeoxycholic 2 ± 1 97* <1 117* 201* <1 189* 15* 230* 235* 63* 7263* 64* 33*
Tau o- sul o- li hocholic 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
Li hocholic 93 ± 27 1 1 14 415 17 16 113 910 148
U sodeoxycholic 65 ± 21 6737* 27645* 96* 3105,873* 1276* 223* 3197* 2725* 23 2812* 2555* 1296*
C27- bile acid species (nM)
Glyco- ihyd oxycholes anoic <1 118 156 189 763 38 1133 24 521 290 59 153 167 27 54
Glyco- dihyd oxycholes anoic <1 15 39 127 117 19 79 861 30 718 33 <1 3
Tau o- ihyd oxycholes anoic 16 ± 3 706 902 1348 4492 553 3199 71 3443 1575 597 2125 1432 194 291
Tau o- dihyd oxycholes anoic <1 87 204 472 708 164 364 14 959 333 77 936 438 69 118
T ihyd oxycholes anoic 4 ± 1 379 32 63 42 17 806 36 30 87 55 14 21 16 46
No e: Con ol samples we e collec ed om 18 heal hy indi iduals.
UDCA- associa ed species we e ma ked wi h an as e isk (*) when he pa ien was ea ed wi h UDCA (12 o 15 mg/kg/day).
Abb e ia ions: ND, no de e mined; UDCA, u sodeoxycholic acid.
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1266
|
RESPONSE OF ACOX2 DEFICIENCY TO UDCA
di ec ed gene ic s udies we e pe o med. The pa ien
showed an inc eased p opo ion o plasma C27- BAs
(Figu e 1) and wo he e ozygous a ian s, p esum-
ably on wo di e en alleles o ACOX2, he c.673C>T
a ian and a dele ion o 4 nucleo ides (c.456_459del)
(Figu e 1). This was p edic ed o gene a e he same
p o ein change (p.Th 154 s) ha had been p e iously
desc ibed o a simila a ian iden i ied in ACOX2 by
Fe dinandusse e al.[10] Li e unc ion es s and anal-
ysis o BA p o ile a e ano he 6 mon hs o UDCA
ea men e ealed main ained no maliza ion o ami-
no ans e ases besides a pe sis en ly al e ed BA p o-
ile (Figu e 1, Table 1, and Table S6). The analysis o
he p oband’s amily showed ha he younge b o he
was homozygous o wild- ype ACOX2, whe eas he
a he was he e ozygous o c.456_459del (p.Th 154 s)
and he mo he showed he c.673C>T (p.A g225T p)
a ian in he e ozygosis (Figu e 1). Thei plasma BA
p o iles we e no mal (Figu e 1, Table S10), wi h C27-
BAs and C24- BAs wi hin no mal anges.
Case 4
A 7- yea - old boy p esen ed a his o y o congeni al hea
disease (mild mi al s enosis, and mode a e- se e e
ao ic insu iciency) wi h sligh de elopmen al delay
and oscilla ing hype ansaminasemia (Table S7). He
was e e ed o he Pedia ic Hepa ology and Li e
T ansplan a ion Uni om Vall d’Heb on Hospi al,
whe e common causes o li e damage we e uled
ou . Abdominal ul asound showed sligh hepa ic hy-
pe echogenici y. Li e biopsy p esen ed sub le po-
lygonal mo phology o he hepa ocy es wi h no o he
ema kable indings (Figu e S2). Due o he mul-
io gan a ec a ion, a comple e exome sequencing
s udy was indica ed. This showed he same double
he e ozygous a ian o ACOX2 as in Case 3; i.e.,
a ian s c.673C>T (p.A g225T p) and c.456_459del
(p.Th 154 s) (Figu e 1). Plasma BA p o iling (Table 1)
con i med enhanced C27- BA p opo ion (Figu e 1).
Diagnosis o ADAH p omp ed UDCA ea men , e-
sul ing in he no maliza ion o amino ans e ase le -
els (Table S7) and a pa ial educ ion in he p opo ion
o C27- BAs in plasma (Figu e 1, Table 1). The anal-
ysis o he p oband’s amily membe s showed ha
he a he ca ied he c.456_459del (p.Th 154 s)
a ian in he e ozygosis and he mo he p esen ed
he c.673C>T (p.A g225T p) a ian in he e ozygo-
sis (Figu e 1). In bo h pa en s, plasma C27- BA and
C24- BA le els we e wi hin no mal anges (Figu e 1;
Table S11).
FIGURE 2 Immunohis ochemis y analysis o acyl- CoA oxidase (ACOX) 2 exp ession in li e biopsies collec ed om pa ien s wi h
ACOX2 de iciency– associa ed hype ansaminasemia (ADAH). Two samples om pa ien s wi h hype ansaminasemia bu no ADAH,
namely, NAFLD F1 and alcoholic ci hosis used as Con ol 1 (A) and Con ol 2 (B), espec i ely. When exp essed, ACOX2 was de ec ed as
in ense g anula s aining inside he hepa ocy es. Nega i e s aining o ACOX2 was seen in li e biopsies om p obands o Case 1 (C), Case
2 (D), Case 3 (E), and Case 4 (F). All images we e ob ained a 20× magni ica ion. Scale ba indica es 50 µm.
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
|
1267
HEPATOLOGY
Immunohis ochemis y s udies
Immunohis ochemis y o li e biopsies showed a com-
ple e absence o ACOX2 s aining in pa ien s wi h ADAH
(Figu e 2). In con as , s aining was posi i e in a se ies
o biopsy samples collec ed om adul pa ien s wi h
di e en li e diseases (da a no shown), wo o which
ha e been included in Figu e 2 as con ols. In con as ,
immunohis ochemis y o ACOX3, ano he acyl- CoA
oxidase ha does no me abolize C27- BA,[10] showed
posi i e signal (wi h di e en in ensi y) in hepa ocy es
in all cases (Figu e S3).
C27- BA– induced oxici y
To analyze he cy o oxic e ec s o THCA, he main
unconjuga ed C27- BA syn hesized in ADAH cases,
we i s analyzed he abili y o THCA o en e li e
cells. CA and GCA up ake by HuH- 7 cells was in-
hibi ed by TCA, which was also aken up by hese
cells. THCA up ake was highe and no a ec ed
by TCA (Figu e 3A). CA, GCA, and TCA up ake by
CHO cells was ma kedly enhanced by NTCP exp es-
sion (Figu e 3B) bu e y mode a ely by OATP1B1
(Figu e 3C) and OATP1B3 (Figu e 3D) exp ession.
In all ci cums ances, a subs an ial THCA up ake was
ound, which was only sligh ly inc eased by NTCP ex-
p ession and was no inhibi ed by TCA; THCA abil-
i y o induce oxida i e s ess was es ed in li e cells.
THCA- induced ROS p oduc ion was highe han ha
induced by C24- BAs, such as CA, chenodeoxycholic
acid (CDCA), and deoxycholic acid (DCA) in HuH- 7
and HuH- 6 cells, whe eas HepG2 and IHH cells
showed esis ance o BA- induced oxida i e s ess
(Figu es 4A– D).
To s udy he e ec on ER s ess, cells sensi i e
(HuH- 7) and esis an (HepG2) o BA- induced oxida i e
s ess we e selec ed. THCA induced a s ong e ec ,
which in mos es s was s onge han ha caused by
C24- BAs (Figu e 4E– K).
The educ ion in iabili y o HepG2 and HuH- 7
cells a e incuba ion wi h THCA was concen a ion-
dependen in bo h cell lines, wi h HepG2 being less
sensi i e han HuH- 7 o THCA- induced cell dea h
(Figu e 4L,M). To con i m he link be ween THCA-
oxici y and he p esence o he p.A g225T p a ian
in ACOX2, monoclonal HuH- 7 cell sublines o e ex-
p essing wild- ype ACOX2 (ACOX2- WT) o he p.A -
g225T p mu a ed a ian (ACOX2- V1) we e gene a ed
(Figu e 5). ACOX2- WT o e exp ession pa ly p o-
ec ed cells om THCA- induced cy o oxici y, whe eas
his C27- BA caused a simila educ ion in cell iabil-
i y in con ol (Mock) and ACOX2- V1 cells (Figu e 4N).
THCA- induced oxida i e s ess was no p e en ed
by coincuba ion wi h UDCA (Figu e 4O). Mo eo e ,
THCA ma kedly up egula ed BAX mRNA, which was
no educed by UDCA (Figu e 4P), whe eas BCL2
was sca cely exp essed by HuH- 7 cells (Figu e 4Q).
Consis en ly, THCA- induced cell dea h was no inhib-
i ed by UDCA (Figu e 4R).
Po en ially ha m ul ACOX2 a ian s
The sea ch o a ian s ha could cause ADAH, ca -
ied ou in he da abases, led o he selec ion o 46
a ian s, o which mos (32 a ian s) had an ex emely
low MAF. Hence, a maximum con ibu ion o 1.56% o
all p edic ed cases o dys unc ional ACOX2 could be
expec ed. Acco dingly, his se was disca ded om
he s udy and a en ion was ocused on he unc ional
analysis o he emaining 14 a ian s (Table 2). This
speci ic se included he c.673C>T (p.A g225T p) a i-
an ini ially iden i ied (ACOX2- V1). Acco ding o SIFT
and PolyPhen- 2 p edic ions, only 11 o hese a ian s
a e expec ed o ha e a unc ional impac on ACOX2
enzyma ic ac i i y. These p edic ions we e u he ali-
da ed by measu ing he abili y o he a ian s, when
exp essed in HuH- 7 cells (Figu e 5A,B), o enhance
THCA bio ans o ma ion in o CA. Monoclonal HuH- 7
cells s ably o e exp essing ACOX2- WT o ACOX2- V1
we e used as posi i e and nega i e con ols, espec-
i ely (Figu e 5C,D). The ac i i y o a ian s V11 and
V14 was simila o ha o ACOX2- WT, sugges ing ha
hese a ian s do no ha e a unc ional impac on en-
zyme ac i i y, as was p edic ed by in silico analysis. In
con as , a ian s V5 and V8 showed a mode a ely de-
c eased ac i i y, whe eas a ian s V2, V4, V6, and V10
we e signi ican ly less unc ional (Figu e 5E).
DISCUSSION
This s udy iden i ied se en indi iduals om ou un-
ela ed amilies ca ying ACOX2 a ian s in ol ed in
ADAH. Among hem, i e indi iduals showed pe sis en
oscilla ing hype ansaminasemia. Th ee pa ien s we e
homozygous ca ie s o c.673C>T (p.A g225T p), he
same a ian ound in he i s ADAH case epo ed,[8]
whe eas he o he wo ca ied he e ozygous c.673C>T
and c.456_459del ACOX2 a ian s in di e en alleles.
In e es ingly, he la e gene a es p.Th 154 s, which e-
sul s in a p ema u e s op codon and a unca ed p o ein.
This change is also caused by ano he simila dele ion
(c.461_464del) in ACOX2 mRNA desc ibed.[10] In con-
as o ou pa ien s, he subjec o ha s udy ca ied he
dele ion in homozygosi y and p esen ed a mo e se e e
condi ion, wi h neu ological symp oms and a ec ion o
lung, li e , hea , and muscle unc ions, and only su -
i ed 6 mon hs.[10] The p esence o o he congeni al
al e a ions esponsible o he mul io gan dys unc ion
o his case was no uled ou . The clinical condi ions
o he six pa ien s wi h ADAH we ha e desc ibed so a
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
1274
|
RESPONSE OF ACOX2 DEFICIENCY TO UDCA
38. Vasa an T, Fe a o E, Ib ahim E, Dixon P, Go elik J, Williamson
C. Hea and bile acids - clinical consequences o al e ed
bile acid me abolism. Biochim Biophys Ac a Mol Basis Dis.
2018;1864(4):1345– 55.
39. Fe dinandusse S, Denis S, Dac emon G, Wande s RJ. Toxici y
o pe oxisomal C27- bile acid in e media es. Mol Gene Me ab.
2009;96(3):121– 8.
SUPPORTING INFORMATION
Addi ional suppo ing in o ma ion can be ound online
in he Suppo ing In o ma ion sec ion a he end o his
a icle.
How o ci e his a icle: Alonso- Peña M,
Espinosa- Escude o R, He aez E, B iz O, Cagigal
ML, Gonzalez- San iago JM, e al. Bene icial e ec
o u sodeoxycholic acid in pa ien s wi h acyl- CoA
oxidase 2 (ACOX2) de iciency– associa ed
hype ansaminasemia. Hepa ology.
2022;76:1259– 1274. h ps://doi.o g/10.1002/
hep.32517
15273350, 2022, 5, Downloaded om h ps://aasldpubs.onlinelib a y.wiley.com/doi/10.1002/hep.32517 by Uni e sidad Del Pais Vasco, Wiley Online Lib a y on [09/02/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
