PROJECT SUMMARY/ABSTRACT Bile flow is essential for normal liver function. Cholestasis is the term for all phenomena where bile flow is im- paired. Cholestatic liver disease results from persistent cholestasis. Substantial progress has been made in un- derstanding of the mechanisms underlying pediatric cholestatic liver disease, including by the current appli- cants. However, in ~30% of children with isolated (primary) cholestasis (PC) the genetic etiology is not identi- fied, despite routine diagnostic sequencing. A closely related clinical presentation results from bile duct injury, and those presenting early in life are described as having neonatal sclerosing cholangitis (NSC). Accurate diag- nosis improves ability to assess disease prognosis and tailor treatment to the underlying disease etiology. This proposal continues the collaboration between Drs. Bull and Thompson, together with the Northwest Genomics Center. The latter provides access to unparalleled resources for genomic data generation and analysis, which can be applied to the unmatched sample and data resources of the 2 largest pediatric liver centers in Europe (King's College London and Children's Memorial Institute in Warsaw), Dr Bull's laboratory, and the NIDDK- funded Childhood Liver Disease Research Network (ChiLDReN). To complement the genetic findings through assessment of disease mechanisms using zebrafish models, Dr. Yin is joining the project. These resources will address the following challenges. In Aim 1, genetic studies will be performed to identify novel genes underlying PC and NSC, and novel mutations in known cholestasis genes. State-of-the-art genetic technologies will be used, including next-generation sequencing of targeted gene panels, exomes, genomes, and RNA, as well as methylation analysis. Available clinical data and tissue collections will be used to find correlates supportive of the causative nature of identified genetic defects, and to establish features associated with mutation in particu- lar genes. In Aim 2, the pathophysiological mechanisms underlying genetic cholestasis will be investigated in zebrafish models. Null-mutant fish have already been generated for usp53 and lsr; orthologues of 2 genes in which mutations have recently been found in children with cholestasis. We will test the hypothesis that Lsr and Usp53 are critical in maintaining the integrity of bile canaliculi. We will assess whether loss of Lsr or Usp53 impairs bile canalicular structure, blood-bile barrier, and apical and basolateral protein trafficking. We will de- termine the interactions of Lsr and Usp53 with other junctional proteins and actin cytoskeleton in hepatocytes. In conclusion, the proposed studies will result in identification of new disease genes and mutations, illuminate commonalities and distinctions between different genetic forms of cholestasis, and will use model systems to shed light on the biological consequences of loss of these genes, which can be difficult to t...