Abstract

Biliary atresia (BA) is a neonatal fibroinflammatory cholangiopathy of infancy and the most common indication for pediatric liver transplantation. We aimed to define the molecular mechanisms responsible for differences in the rate of disease progression among children with BA.

We performed spatial transcriptomics (ST) analysis on frozen liver tissue at transplant from 14 children: BA with survival with native liver (SNL) <2 years (BA1, n=3), BA with SNL >2 years (BA2, n=4), non-BA cholestasis (n=4), and non-diseased donors (n=3). Transcriptional signatures were compared between patient groups by tissue region (scar, hepatocyte, cholangiocyte). Findings were validated in larger patient cohorts that included BA samples at diagnosis.

ST analysis of patients with BA1 showed the most aggressive disease phenotype, characterized by reduced hepatocyte zonation, low expression of homeostatic metabolic signatures, and increased scar heterogeneity enriched for pathways including extracellular matrix remodeling, interferon response, and leukocyte activation. Notably, genes involved in SOX4 hepatocyte-to-cholangiocyte reprogramming were most enriched in patients with BA1. Liver immunohistochemistry with in situ mRNA hybridization showed that patients with BA at diagnosis had increased SOX4 quantification as compared with patients with BA at transplant. Lastly, previously published liver bulk RNA-sequencing data demonstrated higher SOX4 gene-set expression in patients with BA at diagnosis with SNL <2 years.

Children with BA and worse outcomes exhibit increased SOX4 gene-set expression at diagnosis with greater loss of hepatocyte zonation and immune-driven scar heterogeneity at transplant. Further mechanistic studies are needed to determine whether SOX4-associated biliary reprogramming contributes to maladaptive reparative processes in BA.