DAVENPORT 2023 ERH-2022 - 0397.R2 - Proof - Hi

Expert Review of Gastroenterology & Hepatology
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Advances In Understanding of Biliary Atresia Pathogenesis
and Progression – a Riddle Wrapped in a Mystery inside an
Enigma.
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Journal: Expert Review of Gastroenterology & Hepatology

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Manuscript ID ERH-2022--0397.R2
Manuscript Type: Review (Invited)

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biliary atresia, osteopontin, MMP-7, biliary atresia splenic malformation

Keywords:
syndrome, Cat-eye syndrome, liver fibrosis, cytomegalovirus
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URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

Page 1 of 31 Expert Review of Gastroenterology & Hepatology
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3 REVISION #2 Submitted FEB 19th 2012 Expert review in Gastroenterology and Hepatology
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5 Contact: Emma Huck, Commissioning Editor, Expert Review of Gastroenterology & Hepatology
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7 emma.huck@tandf.co.uk
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Advances In Understanding of Biliary Atresia Pathogenesis and
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21 Progression – a Riddle Wrapped in a Mystery inside an Enigma.
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27 Mark Davenport ChM FRCS (Paeds) Professor of Paediatric Surgery
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Rania Kronfli MSc FRCS Consultant Paediatric Surgeon
30 Erica Makin MSc FRCS (Paeds) Consultant Paediatric Surgeon
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34 Dept. of Paediatric Surgery, Kings College Hospital, LONDON
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38 Correspondence:
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Prof M Davenport
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43 Kings College Hospital
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Email: markdav2@ntlworld.com
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Information Classification: General
Expert Review of Gastroenterology & Hepatology Page 2 of 31
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3 ABSTRACT & EXPERT COMMENTARY
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5 Introduction:
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7 Biliary atresia is a potentially fatal condition of the bile ducts – both intra- and extra- for which we
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have no cure. Though principally a cholestatic condition much of its pathology stems from its tendency
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10 to aggressively induce liver fibrosis and ultimately cirrhosis, only partially restrained by
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12 portoenterostomy.
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Areas Covered:
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15 The review is based on current literature exploring the heterogenous nature of biliary atresia. Thus
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17 there are various phenotypes or variants of biliary atresia, each potentially with different aetiological
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backgrounds caused by a number of hypothetical pathological mechanisms thought to be important
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22 Expert Opinion:
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24 Future developments will be made on matching clinical variants with a more distinct
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25 pathophysiological discrimination and those pathways linking the initial cholestatic phase of biliary
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27 atresia to the early stages of fibrosis.
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Search methodology
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33 The review (Oct. - Nov. 2022) is based on a search of PubMed (NLM) using main keyword: “biliary
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35 atresia” with supplementary searches using “fibrosis”; “inflammation”; “BASM”; “genetics”;

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“surgery”; “experimental”; “etiology”; “virology”; “cases”; and “syndromes”.
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40 KEYWORDS: Biliary atresia; biliary atresia splenic malformation syndrome; CMV associated biliary
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42 atresia; liver fibrosis; MMP-7; Osteopontin.
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3 ARTICLE HIGHLIGHTS
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6  Biliary atresia is not a single disease. It has a number of clinically different phenotypes or
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variants (e.g. syndromic; cystic biliary atresia and CMV IgM +ve biliary atresia) in addition to
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9 the most common - “isolated” variant.
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11  It is likely that most variants have an in-utero onset (possibly excepting CMV IgM+ve BA)
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13 with embryological or fetal failure of bile duct development.
14  Underlying aetiological mechanisms may include developmental; genetic; immunological;
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16 and viral factors.
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18  The link and biological mechanism between the clinically obvious cholestasis evident at first
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presentation and the later development of liver fibrosis has not elucidated but remains
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21 crucial to long-term outcome in this disease.
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23  Future progress in untangling this pathophysiological conundrum is awaited to guide future
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applications of adjuvant therapy.
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3 Abbreviations
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5 BASM – Biliary Atresia Splenic Malformation
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7 ICAM – Intercellular Adhesion Molecule
8 Foxp-3 Forkhead box P3
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10 IFN-γ Interferon-Gamma
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12 CABA – Cardiac Associated Biliary Atresia
13 Shh – Sonic hedgehog
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15 TIMP – Tissue Inhibitors of MetalloProteinases
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17 FGF - Fibroblast Growth Factors
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VEGF - Vascular Endothelial Growth Factor

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20 TGF-β - Transforming Growth Factor β -
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22 HIF - Hypoxia-inducible factors

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GPC1 - Glypican1
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25 IL-6, IL-8 etc. Interleukin

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27 CXCL-1 – Chemokine (CXC motif) ligand - 1
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NF-κβ - Nuclear factor – kappa- beta

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30 JAK2/STAT3. Janus kinases (JAKs), signal transducer and activator of transcription (proteins),
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32 MHC – Major Histocompatibility Complex

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3 1.0 Introduction
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5 Biliary atresia (BA) may be pathologicaly defined as obliterative condition of both the intra- and
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7 extrahepatic parts of the biliary tract and may be discerned in the first weeks of life. The usual clinical
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presentation involves a conjugated jaundice; absence of bile within the gastrointestinal tract causing
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10 pale stools, and overspill of the soluble conjugated fraction of bilirubin leading to dark urine.
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12 Cholestasis leads to an aggressive hepatic fibrosis which may well progress to cirrhosis and end-stage
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liver failure. Although a rare disease albeit with major global variation (1 - 2 per 10,000 live births in
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15 Asia [1] and 0.5 – 0.8 per 10,000 in Europe [2] and North America [3], this is the picture that presents
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17 to clinicians throughout the world. What lies behind it in terms of its aetiology and its pathogenesis
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remains veiled in conjecture and speculation. The aim of this chapter is to review these aspects of BA
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22 human condition rather than the various viral-induced mice-based models.

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24 It is certain that BA is not a single disease, in the sense that a number of pathogenic pathways may
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26 have been followed to arrive at the typical scenario described above. A phenotype classification
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28 (Figure 1) can be defined to summarise some of what we observe as clinical BA. Most (~80% in Europe
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29 and North America) are indeed fairly typical in having an atrophic gallbladder, an obliterated
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31 extrahepatic biliary tree (up to and including the portal plate) with a varying degree of liver fibrosis
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33 (the extent of which is time-dependent from birth). The infants, despite the jaundice, are usually
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36 BA as their descriptive term. By contrast, there are infants who have an undoubted syndromic
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38 association. We have defined two of these: the Biliary Atresia Splenic Malformation (BASM) [4,5]
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41 anomalies such as oesophageal and jejunal atresia etc. We will consider the pathogenesis of this group
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43 first.
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45 2.0 Syndromic Biliary Atresia

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48 2.1 Basic Biliary Embryology
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51 The development of the biliary system occurs within two distinct phases and timelines. What will
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53 become the extrahepatic bile duct is identifiable as an outpouching of the foregut at Carnegie embryo
54 stage 11 (4th week) and progresses to a funnel-shaped structure with a gallbladder enveloped by the
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56 primitive liver anlage by Carnegie embryo stage 17 (6th week). Luminal patency appears to be intact
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58 throughout [10, 11].
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3 At the end of this timeline we have the appearance of the hepatoblasts in a predominantly
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5 hematopoietic liver anlage. These start to differentiate into hepatocytes from around 49 days (7th
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7 week), and biliary epithelial cells (now expressing SOX9 and CK19). The latter develops into a two-cell
8 layer around the ingrowing portal venous network and then by a process of selection and deletion
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10 form the “ductal plate” (which can persist in some cases of BA) and then a more mature, tubularising
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12 intrahepatic biliary network.
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At or around 12 weeks gestational age the continuity of extra and intra-hepatic duct network is
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19 This early embryological extrahepatic timeline is shared by determination of visceral situs and
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21 development of the spleen. The definitive portal vein and vena cava occur relatively later in the
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embryo with remodeling involving vitelline and cardinal veins respectively. The co-incidence of all
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24 these somewhat peculiar anomalies points strongly to a time-dependent insult in these early
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26 embryonic weeks. However, the actual mechanism is still somewhat obscure.
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29 It is our belief that the bile duct pathology in Biliary Atresia Splenic Malformation (BASM) infants is
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31 fundamentally one of failure of this extrahepatic bile duct to develop normally. Clinically these infants
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almost invariable have a markedly atrophic gallbladder and absence of the common bile duct with a
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34 largely non-inflammatory porta hepatis.
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2.2 Biliary Atresia Splenic Malformation
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39 Biliary Atresia Splenic Malformation (BASM) syndrome was first defined in 1994 by Davenport et al.
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41 [4], though various components had been recognised earlier (e.g. polysplenia). There is a dramatic
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difference in incidence of BASM between European and American series (10-15%) [3, 7] and various
44 Asian series (<2%) though the reason is obscure [8]. Possibly this might reflect a higher incidence
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46 overall in the Asian series, due largely to the non-syndromic variants.
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48 The phenotype of BASM does vary though should be at least defined by presence of a splenic
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50 malformation – its commonest association. We do not recognise BA and an isolated malrotation as
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52 part of this spectrum, for instance. The splenic malformation is typically polysplenia but sometimes
53 can be asplenia or double spleen [4]. Within the abdominal cavity are other anomalies and include:
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55 situs inversus (30-40%) with and without malrotation; preduodenal portal vein (~40%) and a complete
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57 absence of the intrahepatic vena cava (variable proportion depending on if it is looked for). Cardiac
58 anomalies are also apparent in about half the cases overall with a wide spectrum of cyanotic and non-
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60 cyanotic conditions ranging from severe anomalies such as Tetralogy of Fallot and Transposition of the

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3 Great Vessels to a patent ductus arteriosus and foramen ovale. We recently reviewed our experience
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5 of cardiac anomalies in BA identifying 37 (48%) patients from a series of 524 infants [9]. About 57%
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7 of these cases of Cardiac Associated Biliary Atresia (CABA) either had BASM or were part of the Cat-
8 Eye syndrome [6].
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11 There have been various studies which have tried to link BASM with a variety of genetic mutations.
12 These have included case reports describing mutations in CFC1, NODAL, FOXA2 and ZIC3 [e.g. 12] but
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14 the most recent has been a North America multicentre series of BASM children that used whole exome
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16 screening in 67 infants with the “BASM” phenotype and their parents [13]. This would be a large series
17 for this syndrome but when the study is subject to scrutiny only 60% of this group actually had a splenic
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19 malformation. Other anomalies (e.g. isolated renal anomalies, n=4), that are not really recognised as
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21 being part of the BASM spectrum seem to have been included for some reason. Five children within
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their interpretation of “BASM” had rare biallelic variants in the gene, polycystin 1-like 1 (PKD1L1)
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24 found on chromosome 7.
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27 There may be factors other than genetics that contribute to causation in BASM. The original [4] and
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29 our follow-up [5] BASM series confirmed an association with maternal diabetes. Though this disease
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32 Vessels, spina bifida and sacral agenesis its actual mechanism is still obscure. A recent study in Texas
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34 [3] confirmed this maternal diabetic link us, though intriguingly not only for the BASM infants. The
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35 role of peri-conceptual nutrition and dietary intake in non-BASM infants was studied using the
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37 American National Birth Defects Prevention Study database [14]. This compared 152 (172 isolated BA)
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39 cases and 11,112 controls and described significantly higher values for glycemic index foodstuffs in
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43 2.3 Cat-eye syndrome
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46 The Cat-eye syndrome (CES), aneuploidy of chromosome 22, does have an association with BA [6]. In
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48 our original paper 5 infants (4 with BA) were described with a range of genetic anomalies including
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50 classical Cat-eye syndrome, partial duplication of chromosome 22 (supernumerary der(22) syndrome),
51 and a mosaic for trisomy 22. Clinically these infants showed the typical features of CES, namely
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53 coloboma, cardiac anomalies and anorectal malformations. The nature of the pathogenesis itself is
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3 3.0 Cystic Biliary Atresia
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5 Infants with Cystic Biliary Atresia (CBA) are characterised as having cystic dilatation in an otherwise
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7 obliterated biliary tree and are fairly easily distinguished at laparotomy and cholangiography from
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cystic choledochal malformation (CCM) [15]. Antenatal detection is possible in both though CBA tends
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10 to be smaller. Infants with CCM may be clinically obstructed certainly but in these the intrahepatic bile
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12 ducts are more mature and may dilate. The cholangiogram invariably shows a smooth appearance to
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a “tree-like” structure, whereas the ducts are much more primitive, smaller, irregular and some even
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15 have a “cloud-like” appearance (Figure 2). Bile is found in about 25% of infants with CBA implying
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speculate that in these there is some pathogenic, possibly ischaemic, insult to the distal common bile
20 duct with resultant dilatation later. There are some experimental precedents for this. Lewis Spitz, on
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22 the cusp of becoming Professor of Surgery at Great Ormond Street Hospital, London, completed a
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24 study in six fetal lambs reporting it in 1980. Here he ligated their common bile ducts [16], and noted
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25 that following birth he had created bile-containing extrahepatic cysts similar to the appearance of CBA
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27 in humans. Earlier, in 1969, Pickett and Briggs [17] were able to ligate the hepatic artery in II fetal
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29 lambs with seven of them surviving to term. In these survivors they identified an "interruption of the
30 common duct adjacent to the tie which was found in each case medial to the common duct."
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34 The bile duct (both intra- and extra-) receives its blood supply solely from the hepatic artery and may
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35 certainly be susceptible to some ischaemic event. This concept has been examined by a research group
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37 from Porto Allegre, Brazil. They postulated that ischaemia plays a central role in the cholangiopathy
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39 of non-cystic BA. Thus, Dos Santos et a. [18] used the morphometry of the intrahepatic hepatic
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arterioles and expression of angiogenesis mediators within the liver to show that there was an
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42 increase in arterial medial layer thickness at the time of Kasai portoenterostomy compared to controls.
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44 They also showed that gene expression of hypoxia-inducible factors (HIF), HIF1a and HIF2a, were
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elevated whilst, vascular endothelial growth factors (VEGFA) (VEGFR1 and VEGFR2) were decreased
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47 in those with BA, compared to control livers. Both features seemed to suggest reduced angiogenesis.
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49 It is not clear, however, whether these observations are in any way aetiological or are secondary to
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the onset of structural changes within the liver due to the onset of fibrosis in BA cases.
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54 4.0 Cytomegalovirus IgM +ve Biliary Atresia
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56 For many years the hypothetical role of viruses in this disease has been postulated. This began with
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the REOvirus, took in the animal models using rotavirus but now appear to have coalesced around the
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59 double-stranded DNA virus - cytomegalovirus (CMV). There is certainly excellent evidence of its role
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3 in causing the clinical features of so-called congenital CMV (cCMV) infection which are microcephaly,
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5 neurodevelopmental delay and sensori-neural hearing loss. Indeed, it is the leading non-genetic cause
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7 of the latter and is said to occur in 1–2% of all pregnancies [20].
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9 Something we have defined as CMV IgM+ve BA is much less common and doesn’t have the
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11 neurological sequelae. In 1998, Bjorn Fischler from Stockholm was able to show CMV-IgM antibodies
12 in serum from 38% of BA compared to 6% in age-matched controls [21]. We later showed that this
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14 IgM+ve group had distinct clinical differences (compared to CMV IgM -ve controls) by coming from a
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16 predominantly non-white ethnic background, with a later presentation [22]. This observation has been
17 confirmed in a recent review of four European centres [23]. It is possible to recognise this variant by
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19 its characteristic histological features in the liver at the time of the KPE, showing predominantly
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21 inflammatory features [22]. We further refined this looking at the immunohistochemical
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characteristics of the T cell infiltrate and showed that those with CMV IgM +ve BA had a predominantly
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24 Th-1 T cell infiltrate, compared to both isolated and syndromic forms of BA (Figure 3) [24].
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Though we tend to define our patients on the basis of serology, it should be noted that this may not
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28 be all inclusive. We looked for CMV DNA using real-time PCR in a 67 BA cases, identifying it in 4 CMV-
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30 IgM +ve patients, but also in 2 infants with isolated BA (one negative and one indeterminate serology).
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Furthermore, both these latter cases had high levels of CMV in their urine and “bile” [25].
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34 The actual mechanism of how this injury comes about has been debated for a number of years. The
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simplest explanation is that it is bile duct epithelial injury caused directly by viral action. However,
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37 there is a more eclectic hypothesis involving the induction of an (auto) immune-mediated
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39 proinflammatory state causing secondary cholangiocyte damage. This, therefore, allows perpetuation
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of an innate immune response driven by macrophages, NK cells, Th1 and Th17 cells etc. and is
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In support of this hypothesis, Brindley et al. [26] reported that 56% of BA infants had significant
46 increases in γ-interferon-producing liver T cells in response to CMV exposure compared with a control
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48 group. We looked at plasma aspartate aminotransferase (AST) (as a surrogate marker of active
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50 hepatocyte injury) at presentation in our cohort of IgM +ve BA infants (already higher than controls)
51 and showed a negative correlation with CMV viral load. We interpreted this as that the liver damage
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53 was occurring in the viral clearance phase rather than the earlier stages of the viraemia [27] (Figure
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55 4).
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57 CMV is an uncommon subject for the induction of BA in animals, where Rhesus Rotavirus is much more
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59 commonly used. A group from Fudan University, Shanghai used a guinea-pig model of BA and showed
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3 [28] that there was a predominantly Th1 T cell mediated destructive immune response. While Wen
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5 et al., in a T-reg depleted (anti-CD25 in BALB/c mice) model, showed that low-dose CMV exposure
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7 caused an obvious inflammatory destruction of bile ducts mediated by autoantibodies such as enolase
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10 [29].
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5.0 Isolated Biliary Atresia
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15 This is by far the largest group although that still doesn’t imply complete homogeneity, simply that we
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can’t find any further distinguishing features. It is worthwhile to go through a range of possible
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aetiological mechanisms, examining both their pros and cons, and remembering that most of the
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20 following studies have not really made too many efforts to discriminate on the basis of the foregoing
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clinical separation into variants.
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24 The aforementioned phenotypic classification has been developed on the basis of clinical observation
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26 but there have been some studies involving differential gene expression to support such
27 discrimination. Thus, Pang et al. from Guangdong, China [30] using the BA microarray dataset
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29 GSE46995 and unsupervised cluster analysis in a sample of >50 BA livers identified three distinct
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31 subtypes: “autoimmune”, “viral” and “embryonic”. The first correlated with Fc Gamma Receptor
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32 (FCGR) activation and hub gene FCGR2A and suggested an autoimmune response targeting bile ducts.
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34 The second was associated with immune receptor activity, cytokine receptor, signaling by interleukins,
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36 viral protein interaction suggesting association with viral infection and the third was associated with
37 signaling and regulation of expression of Robo receptors and hub gene ITGB2, corresponding to an
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39 embryonic BA sub-type. Nonetheless, there was no actual attempt to correlate these with the actual
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6.0 Potential Pathways of Pathogenesis
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48 6.1 Genetics Factors
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50 Beyond the genetics of BASM and the syndromic forms there is very little hard data on the role of
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52 genetics in IBA. There may be a genetic predisposition, which in itself implies some kind of “second-
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54 hit” for the full-blown clinical picture. Most of the work on this subject has emerged from varying
55 parts of the world but it is notable that Chinese studies form the bulk of this aspect of research. Garcia-
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57 Barcelo et al. from Hong Kong initially identified mutations in ADD3 (10q25.1-q25.2) in a Han Chinese
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59 population [31]. ADD3 encodes for γ-adducin, which promotes hepatocyte - biliary epithelial cell
60 interconnection and regulates cell migration and contraction of bile cells as a membrane-

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3 cytoskeleton-associated protein. Lam et al. from Hong Kong reported whole-exome screening of 89
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5 non-syndromic “trios”. They extended this concept of genetic influences in BA in another direction by
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7 showing a range of biallelic deleterious variants in “ciliary genes” as expressed in the liver in 31% of
8 their patient [32]. Nonetheless, the degree of risk seems relatively small (odds ratio (OR) [95%
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10 confidence intervals (CI)]= 2.58 [1.15-6.07], p = 0.034). Other predisposing genes have been suggested.
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12 Thus, single nucleotide polymorphisms in mannosidase-1-α-2 (MAN1A2) have recently been
13 associated with BA; though their lack of clinical breakdown precluded real analysis of the relationship
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15 and the quoted OR is still relatively modest [33]. What was interesting but largely based on zebrafish
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17 modelling and cultured cell experiments was its possible role in laterality determination and ciliary
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dysgenesis via its place in the ciliogenesis and planar polarity effector (CPLANE) network of proteins.
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21 In 2010, a group from Philadelphia, PA [34] observed overlapping heterozygous deletions at 2q27.3
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(the location of GPC1), in two unrelated children with BA. GPC1 encodes for glypican1 and this heparin
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24 sulphate proteoglycan modulates various signaling pathways integral to the inflammatory process
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26 such as fibroblast growth factors (FGF), vascular endothelial growth factor A (VEGFA), transforming
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growth factor β (TGF-β) and the hedgehog (Hh) signalling pathway. Later work by the same group [35]
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29 on copy number variants (CNV) in a cohort of 61 BA children and 5,088 controls, showed a significant
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31 increase in deletions at 2q37.3 in the BA group that resulted in deletion of one copy of GPC1.
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36 6.2 Environmental Factors
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38 An intriguing detective story lies behind the discovery of a possible environmental cause for BA [36].
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40 This occurred in a particular place in New South Wales, Australia – the Burunjuk Dam, an area subject
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to periodic drought. During such times, this proved conducive to overgrowth of a particular strain of
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43 the Red Crumbweed (Dysphania glomulifera) around the land revealed by the receding water, on
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45 which pregnant ewes were allowed to feed. The following spring the lambs were afflicted by a BA-
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like condition. Some years after this veterinarian observation, Michael Pack, a biologist from
48 Philadelphia, collected the incriminating vegetation and isolated a group of isoflavonoid compounds
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50 which had specific toxic effects in a Zebrafish model and by extension on the developing ovine biliary
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52 tree. The main one has been labelled as biliatresone [37].
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54 Later work, this time with mice modelling, showed that biliatresone seems to do this by depletion of
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56 glutathione, upregulation of members of the NOTCH and Wnt signaling pathways and downregulation
57 of Sox17 causing disruption of the microtubular structure in cholangiocytes [38,39].
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3 The actual evidence underpinning a role in, humans is much, much less substantial. Dysphania spp,
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5 the original source of biliatresone, is not obviously a normal component of anyone’s diet but some
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7 closely related foodstuffs may be (e.g. chard, sugar beet). There are studies which have sought to look
8 at aspects of nutrition during pregnancy but none convincingly show any real deviation from the norm.
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10 A small American case-control study (n = 152) examined maternal nutrition in detail but really could
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12 not identify much difference [14].
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14 Jiang et al from Shanghai, China [40] widened the search for other potential environmental influences
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16 using a questionnaire-based study looking at demographic differences between a large series of BA
17 patients (n = 613) and matched case-controls. They did find quite a number of differences in the BA
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19 group including a higher incidence of non-Han ethnicity in the parents, lower socio-economic
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21 groupings, older fathers; several pregnancy-related factors such increased drug (illicit and medicinal)
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use, increased consumption of vitamins and supplements, higher incidence of infections (e.g. CMV,
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24 respiratory) and gestational hypertension; and key infant differences including lower birth weights;
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26 prematurity, and more mixed and formula-feeding. Interestingly, gestational diabetes was not more
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prevalent in the BA group. Much of this complements the observations of our own national study
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29 (n=302) published in 2009, which highlighted demographic differences between the IBA and
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31 developmental BA (i.e. BASM, anomalies and cystic BA) such as increased proportion of non-Caucasian
ev
32
ethnicity and breast feeding in the IBA group [2].
33
34
iew
35
36
37 6.3 Biliary atresia and Inflammation
38
39
40 Originally BA was felt to have little in the way of an inflammatory pathogenesis, but during the 1990s
On
41 small case series showed that there were abnormal expression in the liver of the key components of
42
43 the inflammatory cascade. For instance, Peter Dillon from Hershey, PA showed abnormal ICAM
44
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45 expression in six cases [41]. In 2001, we expanded this concept using a whole panoply of antibodies
46 against various components of the inflammatory process in BA livers and bile duct remnants [42].
47
48 We showed that about half expressed MHC Class II antigen; about 30% had a pronounced activated
49
50 mononuclear (CD4+T and NK (CD56) cells)) infiltrate with ICAM and less often VCAM and E-selectin
51
expression on endothelial surfaces. Further study on the various cytokines up to six months post-KPE
52
53 also showed some dramatic elevations (TNF-α, IFN-γ, IL-2, IL-4, IL-18) with signs of resolution if the
54
55 jaundice was resolving [43]. Our latest study further characterised, quantitatively, the nature of the
56
T-cell infiltrate, showing that these were predominantly Th-1 and Th-17, with very little identification
57
58 of Th-2 cells [24]. Kleiman et al. [44] from Hannover confirms a central role for IL-17 with upregulated
59
60 mRNA identified in the liver of their BA patients. Th-17 cells seem to play a critical role in clearing

1
2
3 extracellular pathogens, secreting a host of cytokines (e.g. IL-23, IL-6, IL-8 and IL-1) and chemokines
4
5 (e.g. CXCL (CXC-chemokine ligand)-1 (GRO-a), CXCL2, CXCL3, CXCL6(GCP-2), etc. which mediate tissue
6
7 inflammatory infiltration perhaps by the activation of NF-κβ [45].
8
9 P-selectin (CD 62P) expression has also been shown to be expressed on endothelial surfaces (vascular
10
11 and biliary) compared to cholestatic controls in one Egyptian study (46). They speculate on the
12 implications of this including possible increased recruitment and activation of platelets in the liver.
13
14 How that is related to the thrombocytosis commonly seen in some of these infants (47), is not
15
16 explained.
17
18 T-regs are the anti-inflammatory cells of the host response and there is a hypothesis, though largely
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19
20 built from the experimental viral-mice model, that low levels of T-regs early in the process (?perinatal)
21
might be a key factor in sustaining the inflammatory destruction of cholangiocytes. This was first
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23 proposed in 2012 by Brindley et al. [26] who noted reduced absolute numbers of circulating T-regs
24
compared to controls with a marked association with expression of CMV antibodies. This might
ee
25
26
suggest sequestration, though there was not much evidence for this in lymph nodes though no
27
28 quantification within the liver was done. We found very little accumulation of T-regs in our
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29
30 quantitative study [24]. An alternative explanation is that there is some a functional T-reg deficit with
31
abnormally high methylation of the Foxp3 promotor region of T-regs being shown in BA patients (n =
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32
33 20) in one Chinese study [48] and paralleled observations in their experimental arm using the mouse
34
iew
35 model. Somewhat against this, Yang et al. [49] showed increased liver expression of Foxp3 (T-reg) in
36
the interface areas of the portal tracts of their BA patients, suggesting active recruitment. Most
37
38 recently, it has been appreciated that T-regs are not homogenous and can be divided further on level
39
40 of their ICOS (CD28) expression. ICOS+ Tregs tend to secrete high amounts of IL-10 while ICOS− Tregs
On
41
42
tend to secrete TGF-β1 and are truly suppressive and at least in one study [50] was associated with a
43 better prognosis.
44
ly
45
46 MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by targeting
47 mRNA at the post-transcriptional level and play critical regulatory roles in certain disease states. There
48
49 are a few studies which have looked at this in the context of BA though there are so many individual
50
51 miRNAs (>20,000) that it can be difficult to sort out specific or exclusive patterns
52
53 We have used Next Generation Sequencing (NGS) to investigate miRNA profiles on samples of liver
54
55 and “bile” (obtained at KPE from transected portal plate) in 10 BA infants [25]. This generated 265
56 differentially expressed miRNA. In liver tissues, we identified known miRNA associated with liver and
57
58 bile duct development (e.g. miR-30 and the miR-23 cluster) and those associated with inflammation
59
60 and fibrosis (e.g. miR-29, miR-483, miR-181, miR-199 and miR-200). One striking difference was the

1
2
3 high and consistent expression of miR-486-5p in “bile” samples with relatively low levels in liver tissue.
4
5 Other groups have identified other members. So, Zhao et al. found 23 independent miRNAs to be
6
7 differentially expressed in BA (n = 9) compared to normal controls (n = 6) and highlighted miR-155 as
8 warranting for further study [51]. This they then studied using a variety of experimental models
9
10 showing upregulation of major histocompatibility complex (MHC) I and II and activation of
11
12 JAK2/STAT3.
13
14 Peng et al. investigated them in a diagnostic context in plasma [52] and suggested a couple of
15
16 candidates with high plasma levels (miR-122-5p and miR-100-5p) and others with low levels (e.g. miR-
17 140-3p), though it is difficult to see this as a really discriminatory test given the heterogeneity of the
18
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19 pathogenesis of BA.
20
21
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22
23
24 6.4 Biliary Atresia and Progression to Liver Fibrosis
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25
26 One of the main clinical characteristics of BA which distinguishes it from other neonatal cholestatic
27
28 conditions (e.g. Alagille’s syndrome, α-1-antitrypsin deficiency) is the propensity to progress to early,
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29
30 aggressive fibrosis within the liver leading ultimately to cirrhosis and end-stage liver failure. The chief
31 palliative operation, the Kasai portoenterostomy for BA, may well alleviate cholestasis but, at least in
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32
33 some cases, has no effect on fibrosis leading to ultimate failure.
34
iew
35 Almost certainly there are many mechanisms involved in the genesis of cholestatic, specifically BA,
36
37 liver fibrosis. One of the histological hallmarks of BA is ductular reaction (DR) [53], and these
38
39 proliferating reactive cholangiocytes have the ability to transform and activate myofibroblasts from
40
On
quiescent hepatic stellate cells (HSCs). These express α-smooth muscle actin (α-SMA) produce
41
42 collagen and are promoted by factors such as transforming growth factor (TGF-β1) and platelet-
43
44 derived growth factor (PDGF). Mikko Pakarrinen’s group in a major follow-up study from Helsinki [54]
ly
45 identified increased hepatic gene expression of α-SMA in BA (n = 28) children who nominally had had
46
47 a successful KPE and were jaundice-free. This implied the presence of activated myofibroblasts, long
48
49 after the initial phase of BA. Furthermore, the extent of fibrosis, expansion of the ductular reaction
50
and even the location of α-SMA gene expression correlated. Secretin and its receptor (SCTRA) which
51
52 normally regulates bicarbonate secretion and the creation of a protective “umbrella” protecting
53
54 cholangiocytes from the effects of bile acids also appears to have a role in the DR-fibrosis pathway.
55
Godbole et al. [55] has shown overexpression of SCTR mRNA in proliferating DR cholangiocytes at the
56
57 time of KPE with the higher levels having a diminished native liver survival.
58
59
60

1
2
3 Epithelial-to-mesenchymal transition (EMT) is a pathological process in which round, compact biliary
4
5 epithelial cells become spindle-shaped, losing cell–cell contact with a marked change in surface
6
7 protein expression. So, epithelia proteins such as E-cadherin and CK7 diminish in favour of phenotypic
8 characteristics of mesenchymal cells such as N-cadherin, vimentin, and α-SMA [56, 57]. The process
9
10 appear to be driven by TGF-β1 and inhibited by micro RNA (miR-200b), together with input from the
11
12 Notch, Wnt/β-catenin, and most recently Shh [58] signalling pathways.
13
14 Accumulation of high concentrations of bile acids in the liver and serum are evident in BA [59]. This
15
16 exerts a negative feedback on bile acid synthesis in hepatocytes by down-regulating the rate limiting
17 enzyme in bile acid synthesis cholesterol 7α-hydroxylase (CYP7A1) via farnesoid X-receptor (FXR) and
18
Fo
19 small heterodimer partner (SHP). Previous studies have reported down-regulation of CYP7A1 in BA at
20
21 the time of KPE in relation to non-cholestatic controls [60]. Bile acids also trigger FXR-mediated
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22
Fibroblast Growth Factor 19 (FGF19) production in the liver and small bowel [61] leading to high serum
23
24 levels. Our recent Anglo-Finnish study showed that high levels of serum FGF-19 at the time of KPE
ee
25
26 correlated with serum primary bile acids and had a diminished native liver survival [62].
27
28 The evolution of liver fibrosis post-KPE is a relatively unexplored area simply because so few centres
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29
30 do routine liver biopsies in children and the only ones available are usually explants from those failing
31
and coming to transplant. One exception to this general rule has been Helsinki that do encourage
ev
32
33 routine liver biopsy [63, 64]. In their patients with successful KPE there was diminution of activated
34
iew
35 hepatic stellate cells and liver macrophages; persistence of portal fibroblasts and cholangiocytes with
36
diminution of portal inflammation and ductular reaction with disappearance of periductal α-smooth
37
38 muscle actin expression. There was concomitant up-regulation of key ECM genes (e.g. MMP7, LAMA3
39
40 and ITGB6). Interestingly, this group highlighted a difference in the natural history post-KPE of the
On
41
42
isolated and the syndromic variant with the latter having much less tendency to on-going fibrosis than
43 the former.
44
ly
45
46
47
48 6.5 MMP-7 and Osteopontin
49
50
The matrix metalloproteinase family (MMPs) play a key role in remodelling of the extracellular matrix
51
52 and are produced by connective tissue cells and macrophages. To some extent MMP activity is
53
54 controlled and balanced by Tissue Inhibitors of Metalloproteinases (TIMPs) and in liver fibrosis there
55
is excess accumulation of connective tissue elements, such as collagen.
56
57
58 MMP-7 (initially known as Matrilysin) was discovered in 1988 and consists of 267 amino acids with a
59
zinc core. It is regulated by Wnt/β-catenin signalling reducing cell-to-cell contact by shedding E-
60

1
2
3 cadherin, amplifying inflammation and fibrosis via osteopontin (OPN) and TNF-α. It’s function,
4
5 together with other members of the MMP family, within the liver is degradation of the extracellular
6
7 matrix, specifically Type IV collagen, fibronectin, laminin and elastin [65]. There is a belief that MMP-7
8 is relatively specific to BA, well at least by comparison to other markers of cholestasis during the
9
10 neonatal period. For that reason, it is currently being evaluated as a diagnostic marker in the
11
12 investigation of cholestatic jaundice [66, 67].
13
14 Osteopontin, first identified in 1979, and also known as Bone Sialoprotein-I (BSP-1 or BNSP) overlaps
15
16 much of its actions with members of the MMP family. It is proinflammatory [68] and secreted by a
17 variety of cell types with effects upon the extracellular matrix and is a driver of fibrosis. Whitington et
18
Fo
19 al. [69] showed increased levels of osteopontin in liver tissue obtained from 13 infants with isolated
20
21 BA but interestingly no expression in 4 infants with features of BASM. Immunohistochemical
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22
expression and levels of osteopontin mRNA localized to bile duct epithelia which appeared to be the
23
24 source with a reasonable association with bile duct proliferation and fibrosis. In a pilot study we have
ee
25
26 recently looked at the diagnostic and prognostic value of both serum OPN and serum MMP-7 in BA.
27
Both were consistently higher than age-matched cholestatic controls though discrimination in our
28
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29 series was not as great as previous reports (AUC 0.83 and 0.79 respectively) (Figure 5) [70].
30
31
7.0 Conclusions
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32
33
34 We have presented a current review of the clinical spectrum of BA evident from personal experience
iew
35
36 of over 600 cases. This allowed the development of the variant classification outlined in the text and
37 supported by review of possible causative factors exclusive to that phenotype. The role of genetic
38
39 variation both in the syndromic and isolated forms was reviewed, though the latter has become an
40
On
41 increasingly crowded area with many “susceptibility” genes being identified predominantly in Chinese
42 populations. The inflammatory milieu and an abnormal immune response seems evident in a
43
44 proportion of cases; with abnormal expression of Class II antigens, CAMs and an infiltrate of various T
ly
45
46 cell subsets (Th1, Th17 and less obviously Th2), liberation of inflammatory cytokines (e.g. TNF-α),
47
chemokines (e.g. CXCL-1) and mi-RNAs. Though whether any of this is a primary or secondary event
48
49 is less clear. Current thinking of the role of environmental factors was reviewed though this seems
50
51 unquestioned in certain types of animal BA its relevance to the human condition is much less clear.
52
Finally, we explored the cellular links with and progression to liver fibrosis typified by epithelial to
53
54 mesenchymal transformation, activation of myofibroblasts and the role of MMP-7 and osteopontin.
55
56
57
58
59
60

1
2
3
4
8.0 Expert Opinion
5
6 We have tried to summarise current thinking on various aetiological mechanisms leading to the final
7
8 common pathway recognisable as biliary atresia – this is essentially what aetiological heterogeneity
9
is. There are profound differences of actual pathophysiology within this variant approach, ranging
10
11 from failure in early bile development for the syndromic variants to perinatal inflammatory
12
13 cholangiopathy for that associated with CMV IgM +ve. Much of this stems from clinical observation in
14
large series and supported, after the fact, by very limited range of more fundamental studies into
15
16 genetics and molecular biology for instance. We do have to do more critical analysis of what variant
17
18 we are talking about when trying to discern differences, and one obvious flaw in many outcome
Fo
19
20
studies in clinical series is the lack of CMV testing. We have not reviewed much of the literature based
21 on animal models, which has been principally the rotavirus induced pathology in the BALB/c mice
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22
23 model. A large edifice of biological mechanisms have been constructed using this model but its
24
relevance to human BA may be limited as the only real clinical correlate of this is CMV IgM+ve BA
ee
25
26 which has an incidence of ~10% in European and North American studies. Despite the fantastic analytic
27
28 process that led to the identification of the role of biliatresone in some animal species of BA and
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29
30 supported by proof of its toxicological role in the zebrafish model its actual role in human BA is
31 completely unknown. The problem will be that any evidence of exposure will have disappeared in the
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32
33 mother and the infant long ago.
34
iew
35
36
37
38 We have then explored the actual mechanisms of what makes BA the dangerous disease that it so
39 evidently is which is the cholestasis induced liver fibrosis. Ductular reaction, EMT and the
40
On
41 transformation of hepatic stellate cells into myofibroblasts appear to be key here though what
42
43 specifically initiates or maintains the process is very much unclear. The role and relationships with as
44
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the MMP extended family and the balance with TIMPs; osteopontin, TGF-β, VEGF-A and angiogenesis
45
46 is sketchy at best. Again this may be emblematic of BA that there are so many molecular pathways
47
48 contributing to the laying down of collagen disrupting normal liver architecture.
49
50 Before he became British Prime Minister, Winston S Churchill coined the phrase a “riddle wrapped in
51
52 a mystery, inside an enigma” actually to describe, Russian foreign policy in 1939. This apt epigram
53
might also be applied to some of the aetiological aspects of biliary atresia that we have tried to focus
54
55 on here.
56
57
58
59
60

1
2
3
4
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rP
22 42. Davenport M, Gonde C, Redkar R, Koukoulis G, et al. Immunohistochemistry of the liver and
23
biliary tree in extrahepatic biliary atresia. J Pediatr Surg. 2001; 36: 1017-25. doi:
24
ee
25 10.1053/jpsu.2001.24730.
26
27 43. Narayanaswamy B, Gonde C, Tredger JM, Hussain M, et al. Serial circulating markers of
28
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inflammation in biliary atresia--evolution of the post-operative inflammatory process.

29
30 Hepatology. 2007; 46(1):180-7. doi: 10.1002/hep.21701.
31
ev
32 44. Klemann C, Schröder A, Dreier A, Möhn N et al. Interleukin 17, produced by γδ T Cells,
33
contributes to hepatic inflammation in a mouse model of biliary atresia and is increased in
34
iew
35 livers of patients. Gastroenterology. 2016;150:229-241.e5. doi: 10.1053/j.gastro.2015.09.008.

36
37 45. Lages CS, Simmons J, Maddox A, Jones K, et al. The dendritic cell-T helper 17-macrophage axis
38
39
controls cholangiocyte injury and disease progression in murine and human biliary atresia.
40 Hepatology. 2017; 65:174-188. doi: 10.1002/hep.28851.
On
41
42 46. Sira MM, Sira AM, Ehsan NA, Mosbeh A. P-Selectin (CD62P) Expression in liver tissue of biliary
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44 atresia: a new perspective in etiopathogenesis. J Pediatr Gastroenterol Nutr. 2015
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45 Nov;61(5):561-7. doi: 10.1097/MPG.0000000000000875.

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47 47. Grieve A, Makin E, Davenport M. Aspartate Aminotransferase-to-Platelet ratio index (APRi) in
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49 infants with biliary atresia: prognostic value at presentation. J Pediatr Surg. 2013; 48(4):789-
50 95. doi: 10.1016/j.jpedsurg.2012.10.010.
51
52 48. Li K, Zhang X, Yang L, Wang XX, Yang DH, et al. Foxp3 promoter methylation impairs
53
54 suppressive function of regulatory T cells in biliary atresia. Am J Physiol Gastrointest Liver
55 Physiol. 2016 ; 311(6):G989-G997. doi: 10.1152/ajpgi.00032.2016.
56
57
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60

1
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3 49. Yang Y, Liu YJ, Tang ST, Yang L, et al. Elevated Th17 cells accompanied by decreased regulatory
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5 T cells and cytokine environment in infants with biliary atresia. Pediatr Surg Int. 2013;
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7 29(12):1249-60. doi: 10.1007/s00383-013-3421-6.
8 50. Zhang S, Goswami S, Ma J, Meng L, Wang Y, Zhu F, et al. CD4+T cell subset profiling in biliary
9
10 atresia reveals ICOS- regulatory T cells as a favorable prognostic factor. Front Pediatr. 2019 ;
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12 7:279. doi: 10.3389/fped.2019.00279.
13 51. Zhao R, Dong R, Yang Y, Wang Y, et al. MicroRNA-155 modulates bile duct inflammation by
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15 targeting the suppressor of cytokine signaling 1 in biliary atresia. Pediatr Res. 2017;
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17 82(6):1007-1016. doi: 10.1038/pr.2017.87.
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Fo
52. Peng X, Yang L, Liu H, Pang S, et al. Identification of circulating MicroRNAs in biliary atresia by
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20 Next-Generation Sequencing. J Pediatr Gastroenterol Nutr. 2016; 63(5):518-523. doi:
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22 10.1097/MPG.0000000000001194.
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53. Sato K, Marzioni M, Meng F, Francis H, Glaser S, Alpini G. ductular reaction in liver diseases:
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ee
25 pathological mechanisms and translational significances. Hepatology. 2019 Jan;69(1):420–30.

26
27 54. Kerola A, Lampela H, Lohi J, Heikkilä P, et al. Molecular signature of active fibrogenesis prevails
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in biliary atresia after successful portoenterostomy. Surgery. 2017; 162(3):548-556. doi:

29
30 10.1016/j.surg.2017.04.013.
31
ev
32 55. Godbole N, Nyholm I, Hukkinen M, Davidson JR, et al. Liver secretin receptor predicts
33
portoenterostomy outcomes and liver injury in biliary atresia. Sci Rep. 2022 ; 12(1):7233. doi:
34
iew
35 10.1038/s41598-022-11140-9.
36
37 56. Deng YH, Pu CL, Li YC, Zhu J, et al. Analysis of biliary epithelial–mesenchymal transition in
38
39
portal tract fibrogenesis in biliary atresia. Dig Dis Sci. 2011;56:731–40.
40 57. Xiao, Y.; Wang, J.; Yan, W.; Zhou, Y.; Chen, Y.; Zhou, K.; Wen, J.; Wang, Y.; Cai, W. Dysregulated
On
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42 miR-124 and miR-200 expression contribute to cholangiocyte proliferation in the cholestatic
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44 liver by targeting IL-6/STAT3 signalling. J. Hepatol. 2015, 62, 889–896.
ly
45 58. Siyu P, Junxiang W, Qi W, Yimao Z, Shuguang J. The Role of GLI in the Regulation of Hepatic
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47 Epithelial-Mesenchymal Transition in Biliary Atresia. Front Pediatr. 2022 May 26;10:861826.
48
49 doi: 10.3389/fped.2022.861826.
50 59. Johansson H, Svensson JF, Almström M, van Hul N, et al. Regulation of bile acid metabolism in
51
52 biliary atresia: reduction of FGF19 by Kasai portoenterostomy and possible relation to early
53
54 outcome. Journal of Internal Medicine. 2020;287:534–545.
55 60. Chen HL, Liu YJ, Chen HL, Wu SH, et al. Expression of hepatocyte transporters and nuclear
56
57 receptors in children with early and late-stage biliary atresia. Pediatric Research.
58
59 2008;63:667–673.
60

1
2
3 61. Halilbasic E, Baghdasaryan A, Trauner M. Nuclear receptors as drug targets in cholestatic liver
4
5 diseases. Clinics in Liver Disease. 2013;17:161–189.
6
7 62. Nyholm I, Hukkinen M, Pihlajoki M, Davidson JR et al. Serum Fibroblast Growth Factor 19
8 predicts outcomes of Kasai Portoenterostomy in biliary atresia. Hepatology (2022) (in Press)
9
10 63. Kerola A, Lohi J, Heikkilä P, Mutanen A, Jalanko H, Pakarinen MP. Divergent expression of liver
11
12 transforming growth factor superfamily cytokines after successful portoenterostomy in biliary
13 atresia. Surgery. 2019 May;165(5):905-911. doi: 10.1016/j.surg.2018.12.003.
14
15 64. Kyrönlahti A, Godbole N, Akinrinade O, Soini T, Nyholm I, Andersson N, et al. Evolving up-
16
17 regulation of biliary fibrosis–related extracellular matrix molecules after successful
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portoenterostomy. Hepatology Communications. 2021;5(6):1036–50.

19
20 65. Löffek S, Schilling O, Franzke C-W. Biological role of matrix metalloproteinases: a critical
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22 balance. Eur Resp Journal 2011; 38: 191-208. doi:: 10.1183/09031936.0014651

23
66. Jiang J, Wang J, Shen Z, Lu X, Chen G, et al. Serum MMP-7 in the diagnosis of biliary atresia.
24
ee
25 Pediatrics. 2019; 144(5): e20190902. doi: 10.1542/peds.2019-0902.

26
27 67. Yang L, Zhou Y, Xu PP, Mourya R, Lei HY, et al. Diagnostic accuracy of serum Matrix
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Metalloproteinase-7 for biliary atresia. Hepatology. 2018; 68(6):2069-2077. doi:

29
30 10.1002/hep.30234.
31
ev
32 68. Bezerra JA, Tiao G, Ryckman FC, Alonso M, et al. Genetic induction of proinflammatory
33
immunity in children with biliary atresia. Lancet. 2002; 360(9346):1653–9. doi:
34
iew
35 10.1016/S0140-6736(02)11603-5
36
37 69. Whitington PF, Malladi P, Melin-Aldana H, Azzam R, Mack CL, Sahai et al. Expression of
38
39
osteopontin correlates with portal biliary proliferation and fibrosis in biliary atresia. Pediatr.
40 Res. 2005, 57, 837–844.
On
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42 70. Aldeiri B, Si T, Huang Z, Torner N, et al. Matrix metalloproteinase-7 and osteopontin serum
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44 levels as biomarkers for biliary atresia. In Press J Pediatr Gastrol Nutr 2023
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Selected References
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6
7 **
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1. Davenport M, Tizzard SA, Underhill J, Mieli-Vergani G, Portmann B, Hadžić N. The biliary
10
11 atresia splenic malformation syndrome: A 28-year single-center retrospective study. J
12
13 Pediatr. 2006;149(3):393–400. [57 citations]
14
15 Aside from isolated BA, BASM is the commonest variant (~10% in European and North American
16
17 series). This paper is the follow-up paper to the original report in 1993 and defines the range of
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19 non-hepatobiliary features (e.g. situs inversus) in the syndrome while establishing its relationship
20 with maternal diabetes.
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22
23 2. Berauer JP, Mezina AI, Okou DT, Sabo A, Muzny DM, Gibbs RA, et al. Identification of
24 Polycystic Kidney Disease 1 Like 1 Gene Variants in children with Biliary Atresia Splenic
ee
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26 Malformation Syndrome. Hepatology. 2019;70(3):899–910. [21 citations]
27
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First systematic study using whole exome screening of a cohort of children with “BASM” and their
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30 parents.
31
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32 3. Lorent K, Gong W, Koo KA, Waisbourd-Zinman O, et al. Identification of a plant isoflavonoid

33
34 that causes biliary atresia. Sci Transl Med. 2015 May 6;7(286):286ra67. doi:
iew
35
36 10.1126/scitranslmed.aaa1652. PMID: 25947162; PMCID: PMC4784984 [53 citations]
37
38 Relates the story of the origins and identification of the isoflavonoid biliatresone, from its source
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40 in plants (Dysphania spp.) to its identification using the zebrafish model.
On
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42
43
44
ly
45 *
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48 4. Brindley SM, Lanham AM, Karrer FM, Tucker RM, Fontenot AP, Mack CL. Cytomegalovirus-
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specific T-cell reactivity in biliary atresia at the time of diagnosis is associated with deficits in
50
51 regulatory T cells. Hepatology. 2012;55(4):1130–8.
52
53
A study identifying an unexpectedly high proportion (>50%) of North American infants with
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55 infiltrating IFN-γ T cells that on cell culture studies showed a specific response to CMV and a
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57 positive correlation with levels of plasma CMV IgM.
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60

1
2
3 5. Narayanaswamy B, Gonde C, Tredger JM, Hussain M, et al. Serial circulating markers of
4
5 inflammation in biliary atresia--evolution of the post-operative inflammatory process.
6
7 Hepatology. 2007; 46(1):180-7. doi: 10.1002/hep.21701. [38 citations]
8
9 Key paper identifying the cytokine profile of the inflammatory process at time of presentation
10
11 and its prolonged progression phase thereafter.
12
13 6. Fischler B, Ehrnst A, Forsgren M, Örvell C, Nemeth A. The viral association of neonatal
14
15 cholestasis in Sweden: a possible link between cytomegalovirus infection and extrahepatic
16 biliary atresia. J Pediatr Gastroenterol Nutr 1998; 27: 57–64. [55 citations]
17
18
Fo
19 An early observational study showing an unusually high prevalence of cytomegalovirus

20
antibodies in infants with BA (together with their mothers).
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22
23 7. Zani A, Quaglia A, Hadzic N, Zuckerman M, Davenport M. Cytomegalovirus-associated biliary
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atresia: An aetiological and prognostic subgroup. J. Pediatr Surg. 2015, 50, 1739–1745. [56
ee
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26 citations]
27
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Large scale comparative study of CMV IgM +ve and -ve infants showing key differences in
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30 ethnicity, time of presentation, liver histology and importantly outcome following
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32 portoenterostomy.
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6 FIGURES
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Figure 1: Schematic illustration of “Aetiological Heterogeneity”.
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11 Figure 2: Cystic biliary atresia: (A) on-table appearance of antenatally-detected cyst (white arrows),
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atrophic and solid gallbladder (black arrows) with cholangiogram catheter in-situ. (B) radiological
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14 appearance of cyst and primitive intrahepatic biliary ductules (circled).
15
16
Figure 3: Portal tract T cell subset quantification (Th-1 [A] and Th-17 [B]) according to diagnostic
17
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category: CMV IgM + ve BA (n = 9); BASM (n = 9); isolated BA (n = 19). [reproduced with permission
19
20 from [24]].
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22 Figure 4: Relationship of aspartate aminotransferase (AST) levels with viral load in CMV +ve Biliary
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24 Atresia (n = 31), showing inverse correlation (rS = − 0.59; P = 0.002). [Reproduced with permission
ee
25
26
from [27]].
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28 Figure 5: Receiver operator curve (ROC) in the detection of BA using pre KPE serum value of MMP7
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30 (A) and OPN (B). AUC 0.83 and 0.79 respectively [reproduced with permission from [70]].
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27 338x190mm (96 x 96 DPI)

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31 Cystic biliary atresia: (A) on-table appearance of antenatally-detected cyst (white arrows), atrophic and
32 solid gallbladder (black arrows) with cholangiogram catheter in-situ. (B) radiological appearance of cyst and
iew
primitive intrahepatic biliary ductules (circled).

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46 CMV IgM + ve BA (n = 9); BASM (n = 9); isolated BA (n = 19). [reproduced with permission from [24]].
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Expert Review of Gastroenterology & Hepatology

Journal: Expert Review of Gastroenterology & Hepatology

Manuscript Type: Review (Invited)

biliary atresia, osteopontin, MMP-7, biliary atresia splenic malformation

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

41 Dept. Paed Surgery

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35 atresia” with supplementary searches using “fibrosis”; “inflammation”; “BASM”; “genetics”;

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VEGF - Vascular Endothelial Growth Factor

22 HIF - Hypoxia-inducible factors

25 IL-6, IL-8 etc. Interleukin

NF-κβ - Nuclear factor – kappa- beta

32 MHC – Major Histocompatibility Complex

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22 human condition rather than the various viral-induced mice-based models.

45 2.0 Syndromic Biliary Atresia

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the isolated BA group (OR = 1.25).

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41 clinical phenotype of the cases themselves.

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41 (CABA): a prognostic subgroup. Arch Dis Child. 2021 Jan;106(1):68-72. doi:

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sheep. J Pediatr Surg. 1969; 4: 95

35 biliary atresia. J Pediatr Gastroenterol Nutr 1998; 27: 57–64.

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

22 neonatal mice. Lab Invest. 2015; 95(2):180-92. doi: 10.1038/labinvest.2014.148.

32 25. doi: 10.1093/hmg/ddq196

35 mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel

45 suggest region of potential disease susceptibility in 2q37.3. Am J Med Genet A. 2010;

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

inflammation in biliary atresia--evolution of the post-operative inflammatory process.

35 livers of patients. Gastroenterology. 2016;150:229-241.e5. doi: 10.1053/j.gastro.2015.09.008.

45 Nov;61(5):561-7. doi: 10.1097/MPG.0000000000000875.

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

25 pathological mechanisms and translational significances. Hepatology. 2019 Jan;69(1):420–30.

in biliary atresia after successful portoenterostomy. Surgery. 2017; 162(3):548-556. doi:

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

portoenterostomy. Hepatology Communications. 2021;5(6):1036–50.

22 balance. Eur Resp Journal 2011; 38: 191-208. doi:: 10.1183/09031936.0014651

25 Pediatrics. 2019; 144(5): e20190902. doi: 10.1542/peds.2019-0902.

Metalloproteinase-7 for biliary atresia. Hepatology. 2018; 68(6):2069-2077. doi:

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

32 3. Lorent K, Gong W, Koo KA, Waisbourd-Zinman O, et al. Identification of a plant isoflavonoid

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

19 An early observational study showing an unusually high prevalence of cytomegalovirus

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

URL: https://mc.manuscriptcentral.com/erh Email: IERH-peerreview@journals.tandf.co.uk

27 338x190mm (96 x 96 DPI)

primitive intrahepatic biliary ductules (circled).