Objectives and Study

Recent single-cell transcriptomic data has implicated foetal immune cells in the pathogenesis of biliary atresia (BA) but why there are immature B-cells present in the postnatal liver is unknown. To understand the mechanisms behind this, we performed a meta-analysis of RNA sequencing data from infants with BA at the time of Kasai and age-matched controls. Previous analyses had been limited by small numbers of controls; however, here, we have increased power to detect associations.

Methods

We obtained transcriptomic data from n=177 children with BA and n=78 controls from Gene Expression Omnibus. After filtering and quality control, we performed differential gene expression analysis comparing BA versus control (using DESeq2), with p-value adjustment for multiple testing.

Results

We identified 1,815 significantly differentially expressed genes (Fig. 1A). Many were involved in extracellular matrix remodelling/fibrosis (e.g. CAPG, TGFBI) and phagocytic activity (e.g. CHIT1, CYBA). We observed increased expression of TREM2 (Fig. 1B), which is implicated in profibrogenic scar-associated macrophages in adults and has not previously been described in children.

Patients with BA had increased expression of multiple markers of haematopoiesis, including erythroid lineage (e.g. GYPA, HBA1) and early B-cell (e.g. RAG2) (Fig. 1C). We identified up-regulation of insulin-like growth factor 2 (IGF2, log2 fold change 2.5, pFDR = 2.8x10-40, Fig. 1D). IGF2 is a growth factor for haematopoietic stem cells in the foetal liver. IGF2 is secreted by hepatocyte progenitors and we observed upregulation of markers of these progenitors (e.g. DLK1, Fig. 1E).

Conclusions

Postnatal liver haematopoiesis is active in children with BA but not controls. Ongoing haematopoiesis may be driven by IGF2 secreted by hepatocyte progenitors. In contrast to other causes of neonatal hepatitis, specific interactions between haematopoiesis and other immune cells may play a role in exacerbating intra-hepatic injury in BA.

Objectives and Study

Patients with progressive familial intrahepatic cholestasis (PFIC) may have continued hepatic damage leading to liver transplantation (LT). The efficacy and safety of odevixibat, an ileal bile acid transporter inhibitor, were assessed in patients with PFIC in the phase 3 PEDFIC 1 and PEDFIC 2 studies. In a pooled analysis of data from these studies, we analysed native liver survival (NLS) in odevixibat-treated patients who met serum bile acid (sBA) treatment response criteria (sBAs reduced ≥70% or levels ≤70 µmol/L at 6 months). NLS was also analysed in partial sBA responders (patients with sBA reductions ≥30% to <70% at 6 months) and nonresponders (patients with sBA reductions <30% at 6 months or who underwent LT or discontinued treatment before 6 months).

Methods

PEDFIC 1 (NCT03566238) was a 24-week, randomised, placebo-controlled study in children with PFIC1 or PFIC2. PEDFIC 2 (NCT03659916) is an ongoing 72‑week extension study in patients of any age with any PFIC type. This pooled analysis spans from patients’ first dose of odevixibat to a cut-off date of 31 January 2022.

Results

Of 98 patients analysed (mean treatment duration, 88 weeks), 35 (36%) were sBA responders, 14 (14%) were partial sBA responders, and 49 (50%) were nonresponders. Mean sBA reductions at 6 months were 87% in responders and 44% in partial responders; there was a mean increase of 27% in nonresponders. All 35 sBA responders and 13 of the 14 partial sBA responders remained transplant free; 8 of the 49 nonresponders underwent LT (Figure). sBA responders had mean improvements at week 24 of treatment vs baseline in alanine aminotransferase and total bilirubin levels.

Conclusions

sBA decreases at 6 months were strongly associated with NLS for up to 3 years in odevixibat-treated patients with PFIC.

Objectives and Study

Monogenic liver disease is caused by rare, pathogenic mutations. Exome and genome sequencing frequently identifies variants of unknown significance in these genes. It is not clear whether such non-pathogenic variants confer increased risk of liver injury beyond childhood. Here, we found that these variants increase the severity of liver damage and may act as a ‘second hit’.

Methods

We identified 77 monogenic paediatric liver diseases. For each gene, we searched for evidence of a liver phenotype in individuals not known to have genetic disease using population-based datasets. We identified genome-wide significant associations (p<5x10-8) between variants (e.g. single nucleotide polymorphisms) and liver biochemistry (ALT, bilirubin, GGT, ALP) in n=1,654,950 participants from the Common Metabolic Disease Portal and n=394,841 from UK BioBank using GeneBass.

Results

We found 89 genome-wide associations for biomarkers of liver injury in otherwise apparently healthy individuals across genes from 44/77 (57%) monogenic disorders (Fig 1). For example, common variants in ABCB11 (the cause of PFIC type 2) were associated with GGT (p=2.0x10-33) and ALT (p=8.4x10-39). Similarly, common polymorphisms in JAG1 (that do not cause Alagille’s syndrome) were associated with GGT (p=2.3x10-9) and ALT (p=5.9x10-10).

Significant associations were found most frequently for 5/7 (71%) of cholestatic disorders and 5/7 (71%) bile acid metabolism disorders, compared to 3/8 (38%) of congenital fibrotic disorders.

In addition to affecting liver enzymes, serum lipid profile (e.g. total cholesterol) was affected by genes from 23/44 (52%).

Conclusions

Common variants in genes that cause rare monogenic liver disease also confer a risk of liver injury later in life. Understanding the mechanisms of these genes provides an opportunity for recognition and treatment of common liver diseases.

Objectives and Study

Background: Bacterial translocation (BT), and Pathogen Associated Molecular Patterns (PAMPS), propagate a pro-inflammatory response in chronic liver diseases. However, the role of BT, the immune system, and their interaction, is as-yet incompletely characterised in Biliary Atresia (BA).

Aims: To characterise the association of immune markers in BA, before and after Kasai portoenterostomy (KPE), with fibrosis and BT, and clinical outcomes .

Methods

Plasma samples were prospectively collected from BA infants (n=55) pre-KPE, 6weeks-, 3months-, and 6months-post-KPE. Th1-(IL-2,IFNy), Th2-(IL-4,IL-10), TH17-(IL-17,1Lβ-23), macrophage-associated (IL-6, IL-8, TNFα, IL-1β) cytokines, and cellular adhesion molecules (ICAM-1,VCAM-1), BT biomarkers (LBP and D-lactate) were measured. Clinical outcomes: 6month-JC (jaundice clearance), 1year-LT (liver transplantation), cholangitis by 6month-post-KPE, fibrosis biomarkers [Aspartate Aminotransferase-to-platelet ratio index (APRi); Liver Stiffness Measurement (LSM)]. Additionally, we investigated the course of the assessed immune markers via longitudinal analysis, using generalised estimating equations

Results

Pre-KPE, immune markers were similar between clinical outcome groups. By 6weeks-post-KPE, increased IL-4, IL-8, IL-1β and ICAM-1 were associated with 1-year-LT. ICAM-1 was associated with poor 6month-JC and with fibrosis biomarkers [APRi;rs=0.6,p<0.001:LSM ;rs=0.6,p<0.001]. By 3months-post-KPE, further increases in macrophage-associated cytokines in non-favourable outcome groups (1year-LT and poor 6month-JC), were evident. VCAM-1/ICAM-1, IL-8 and IL-1β positively correlated with APRi and LSM. We found an increased rise of IL-17 [1.14ng/L/month], IFN-y [4.04ng/L/month] ICAM-1 [76 ug/L/month] and VCAM-1 [142ug/L/month] over the 6month-period, in 1yr-LT group. IL-17, 6weeks-post-KPE, associated with cholangitis [p=0.03]. At early-post-KPE time-points, macrophage markers (IL-6, IL-8, TNFa) and ICAM-1/VCAM-1 positively correlated with LBP levels; macrophage markers/TH2/Th17 and adhesion molecules, positively correlated with D-lactate.

Conclusions

Divergence of innate (macrophage) and adaptive (Th1/Th2/Th17) immune pathways early post-KPE, strongly discriminates BA infants with non-favourable clinical outcomes. This novel data provides convincing evidence for BT in macrophage activation, upregulation of adhesion molecules, and fibrosis. Consequently, targeted microbiota-modulatory therapy, early-post-KPE, could dampen PAMP-associated damage in BA.

Objectives and Study

miR-29a reveals and influences numerous physiological phenomena. Of note, miR-29a accessible ameliorates high-fat diet (HFD)-induced liver dysfunctions in mice. However, the miR-29aTg effect on gut microbiome composition and HFD-induced dysbiosis in metabolic disturbance remains unclear. Here, we provided compelling evidence for the protection role of miR-29a in between gut barrier dysfunction and steatohepatitis in mice.

Methods

We bred miR-29a transgenic mice (miR-29aTg) to characterize features of intestinal, serum biochemical, and feces microbiota profiling compared with WT (wild-type) mice. Mice were fed HFD for 8 weeks to induce non-alcoholic fatty liver disease (NAFLD), and intestinal dysfunction was determined via histopathological analysis.

Results

miR-29aTg shape better lipid metabolism capability that decreased total cholesterol and triglyceride in serum than the WT group of the same age. We further demonstrated that miR-29aTg contributed to advanced intestinal integrity by maintaining periodic acid Schiff positive cell numbers and variation of feces microorganisms. HFD-induced bacterial community disturbance and NAFLD resulted in more severe WT than miR-29aTg. Mechanistically, microorganism profiling exhibited Lactobacillus, Ruminiclostridium_9, and Lachnoclostridium enrichment in miR-29aTg and significantly decreased interleukin-6 expression in the liver and intestinal tract.

Conclusions

This study provides new evidence shedding light on the host genetic background of miR-29a that protects against NAFLD and other intestinal disorders.

Objectives and Study

The adenosine triphosphate (ATP) binding cassette subfamily B member 4 (ABCB4) gene encodes for the multi drug resistance 3 protein (MDR3) which translocates phosphatidylcholine (PC) across the canalicular membrane of the hepatocyte. Defects in MDR3 is associated with progressive familiar intrahepatic cholestasis type 3 (PFIC 3). In this research, we sought to determine the contribution of MDR3 heterozygous variants in a large cohort of infants and children with cholestatic liver disease.

Methods

All children seen in the Paediatric Liver, GI and Nutrition Centre at King’s College Hospital, London between 2004 to 2021 with heterozygous status for an ABCB4 variant were included in the study. Patients’ files and electronic health records were reviewed for clinical, laboratory, histological and radiological information.

Results

Ninety-two children were identified with a monoallelic variant in ABCB4 with an average age at presentation with cholestasis of 4 years and 8 months (minimum 1 week, maximum 15 years). The presenting problem differed according to the age in which they presented and included infantile jaundice (n = 52; 57%), gallstones (n = 9; 10%), cholestatic hepatitis (n = 8; 9%) and pruritus (n = 5; 6%). Thirty-two genetic variants were identified in 92 patients including 23 newly described variants and 19 variants which were considered deleterious using in silico prediction tools including 9 missense, 2 in-frame, 2 frameshift, 5 splice site / splice region and 1 termination variants. At average follow up after 20 months, gamma glutamyl-transferase levels and bile duct dilation on imaging tended to normalise.

Conclusions

Rare variants in MDR3 were overrepresented in this cohort providing insight that ABCB4 heterozygosity contributes to cholestatic disease in children. However, the clinical phenotype was highly variable and the overall trend was towards spontaneous biochemical and radiological normalisation.

Objectives and Study

Patients with biliary atresia (BA) and ongoing cytomegalovirus (CMV) infection may have poorer outcomes after Kasai portoenterostomy (KPE) than uninfected patients, but there is no consensus on the usefulness of viral testing and antiviral treatment (AVT). We aimed to investigate the management of CMV infection after KPE in patients with BA.

Methods

An online survey with 10 multiple choice questions was offered to participants in an international congress on BA, organised in collaboration with European Reference Network (ERN) for rare liver diseases in 2022. Answers to questions were either dichotomic or in intervals for numbers. Ongoing CMV infection was defined by the detection of CMV-IgM in serum and/or CMV-DNA by PCR in blood and/or urine.

Results

There were 43 respondents (24 pediatric gastroenterologists/hepatologists, 17 pediatric surgeons, and 2 transplant surgeons) from 36 centres in 26 different countries globally.

The total number of BA patients per year was in the interval of 208-380, centres reported between 0-5 to > 20 patients per year (median 6-10). Testing for CMV infection was performed in 27 centres (75%), of which 18 (67%) would give AVT. Differece in incidence of CMV infection is shown in figure 1.

Willingness to treat the infection did not differ between centres with low and high CMV incidence. Surgeons tended to be more likely than gastroenterologist/hepatologists to test for CMV infection (p = 0.11). Conversely, gastroenterologist/hepatologists were more likely to treat the infection (p = 0.12). Clinicians from 33 centres expressed an interest in a future randomized study.

Conclusions

- A majority of centres test for CMV and a considerable proportion give antiviral treatment, this despite the lack of solid evidence on its benefits.

- Our data suggest that a randomized multicentre controlled treatment study is both needed and feasible, utilizing networks such as ERN Rare Liver and ESPGHAN.

Objectives and Study

Bile Salt Export Pump (BSEP) deficiency, or PFIC2, is a rare genetic liver disease. It often leads to end-stage cholestatic liver disease and need for transplantation. The p.E297G missense mutation with suggested residual BSEP function is associated with a milder course of disease and delayed need for transplantation. BSEP knock-out mouse models exist, but are unfit to study interventions aiming to improve BSEP function, due to complete absence of BSEP protein. No in vivo models with milder mutations are available. Therefore, we aimed to generate a p.E297G BSEP knock-in (KI) mouse model.

Methods

The KI model was generated by CRISPR-Cas9 technology. Female and male KI mice (n=8, each) and WT littermates (n=7, each) were fed chow diet, terminated at age 14 weeks and characterized for BSEP expression and liver pathology. The BSEP transport capacity of was determined in vivo by measuring bile flow during IV administration of increasing dosages of the bile acid tauroursodeoxycholic acid (TUDCA).

Results

Western blotting showed reduced BSEP protein expression in KI vs. WT livers, due to impaired BSEP glycosylation. The capacity to induce bile flow upon TUDCA administration was strongly reduced in KI mice (Figure). Median liver weight was increased in KI vs. WT mice (Females: 2.1g vs. 1.0g; Males: 2.3g vs 1.4g; each P<0.001). Median plasma AST levels were also significantly increased in KI vs. WT mice (Females: 187 vs. 50 µmol/l; Males: 198 vs. 60 µmol/l , P=0.01 and P<0.001; resp.), as were median plasma ALT levels (Females: 160µmol/l vs. 25µmol/l; Males: 248µmol/l KI vs. 30µmol/l; P=0.02 and P<0.001; resp.). Histological analysis showed mild fibrosis in KI livers.

Conclusions

The p.E297G mouse model displays marked liver pathology and provides a new model for BSEP deficiency with suggested residual BSEP function. We anticipate its importance for future in vivo testing of medications that stimulate residual BSEP functionality.