Bile flow is essential for normal liver function. Cholestasis is the term for all phenomena where bile flow is impaired. Over the last few years, great progress has been made in our understanding of the mechanisms underlying pediatric cholestatic liver disease, including by the current applicants. Despite this, in 40% of children, the primary genetic etiology of cholestasis has not been identified, after targeted next-generation sequencing (tNGS) of known cholestasis genes. Furthermore, our understanding of these diseases, and our ability to treat them, is still limited. This proposal continues the collaboration between Drs. Bull and Thompson. It brings together 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 NIH-funded Childhood Liver Disease Research Network (ChiLDReN). This collection of resources and experience will enable the following challenges to be addressed.
In Aim 1, genetic studies will be performed to identify novel cholestasis genes, and novel mutations in known cholestasis genes.
This aim will be accomplished using state-of-the-art genetic technologies including: tNGS, whole exome sequence (WES), whole genome sequencing, RNA-sequencing and methylation analysis.
In Aim 2, we will characterize the features of genetically distinct forms of cholestasis, through assessment of clinical and biochemical data from patients. Extensive tissue collections are available and histological and immunohistochemical analysis will be undertaken.
In Aim 3, we will investigate the pathophysiological mechanisms underlying genetic cholestasis. Animal models of known genetic forms of cholestasis typically fail to replicate human phenotypes well, so in vitro model systems are required to investigate disease mechanisms and test potential treatments. In vitro systems may be used to assess promoter function, splicing, trafficking and membrane transport, as appropriate. Moreover, CRISPR/Cas9 technology will be used to introduce homozygous mutations into induced pluripotent stem cells (iPSCs), to generate mutated hepatocyte and cholangiocyte-like cells. These cells will be used to explore the consequences of mutations in disease-causing genes, initially focused on increased understanding of disease mechanism in TJP2 deficiency. Preliminary data have shown these cells form canaliculi with tight junctions between cells. Use of such cells will allow us, for example, to test the permeability of tight junctions in vitro. In conclusion, these proposed studies will result in identification of new disease genes and mutations, illuminate the commonalities and differences between different genetic forms of cholestasis, and shed light on basic physiology and disease mechanisms.
These aims fulfill key goals of ChiLDReN and are central to the mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), as stated in its Action Plan for Liver Disease Research.
/ PUBLIC HEALTH RELEVANCE Fifteen children are diagnosed with liver disease each day in the US. Last year 580 children received a liver transplant in this country, and another 24 died whilst awaiting one. The great majority of these transplants were for cholestatic liver disease. This study is aimed at understanding the genetic and biological causes of cholestatic liver disease in children. Gaining this knowledge is a vital step in improving diagnosis and targeting treatment.
| Bull, Laura N; Pawlikowska, Ludmila; Strautnieks, Sandra et al. (2018) Outcomes of surgical management of familial intrahepatic cholestasis 1 and bile salt export protein deficiencies. Hepatol Commun 2:515-528 |
| Grammatikopoulos, Tassos; Sambrotta, Melissa; Strautnieks, Sandra et al. (2016) Mutations in DCDC2 (doublecortin domain containing protein 2) in neonatal sclerosing cholangitis. J Hepatol 65:1179-1187 |
| Zhou, Shengmei; Hertel, Paula M; Finegold, Milton J et al. (2015) Hepatocellular carcinoma associated with tight-junction protein 2 deficiency. Hepatology 62:1914-6 |
| Sambrotta, Melissa; Strautnieks, Sandra; Papouli, Efterpi et al. (2014) Mutations in TJP2 cause progressive cholestatic liver disease. Nat Genet 46:326-8 |
