Before birth many functions required of the liver can be replaced by the placenta/mother. It is therefore in the first few months of life that the majoriy of congenital or genetic liver disease will present. The great majority of these diseases are cholestatic diseases, in which there is failure to make, or drain, bile from the liver into the intestine. A clinically defined group of patients was identified some years ago, and their disorder termed progressive familial intrahepatic cholestasis (PFIC). Significant advances in the understanding of the genetic basis of PFIC and related disorders have been made by the current applicants. However, we know that between one third and one half of these patients remain without a precise genetic diagnosis. This is partly due to limitations in the currently employed diagnostic pathways, which are expensive and lengthy;however, it is due to a greater degree to our not having yet discovered all the causative genes. In this proposal, we bring together sample resources from the NIH-funded Childhood Liver Disease Research and Education Network (ChiLDREN), Dr. Bull's laboratory, and the 2 largest pediatric liver centers in Europe. By combining these sample resources and our experience in the field, with recent dramatic advances in genetic technology, we are poised to achieve the goals outlined here.
Aim 1 of the current study will implement cost-effective, custom- designed next-generation sequencing assays, and supporting techniques, to screen patients from our large sample resource for mutation in key known cholestasis genes (i.e. genes mutated in forms of cholestasis). In so doing, we will define a cohort of patients without detected mutation, who are therefore suitable for inclusion in studies aimed at new gene discovery.
Aim 2 A will identify novel cholestasis genes and mutations using a powerful recently developed technique, termed 'whole-exome'sequencing. This technique, in which coding sequence of all known genes is sequenced, is remarkably efficient and cost-effective. Exome sequence of a subset of patients will be generated and analyzed to identify mutations and candidate disease genes, and results confirmed in a larger patient set. The data gained from the gene discovery components of the study will be used to refine a diagnostic testing pathway in an iterative fashion.
Aim 2 B will investigate some newly identified genes and variants to assess their functional roles and consequences. Transcript structure and levels, protein levels and localization, as well as ultrastructural features, may be assessed. Mutations in regulatory elements may be assessed using in vitro assays. Patient liver tissue and appropriate cell lines will be used. The result of ur proposed studies will be identification of new genes and mutations involved in these diseases, which will inevitably shed light on basic physiology, and lead to development of robust, rapid and affordable diagnostic testing. These goals are central to both the strategy of the Childhood Liver Disease Research and Education Network (ChiLDREN) and 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 525 children received a liver transplant in this country, and another 62 died whilst awaiting one. The great majority of these transplants were for cholestatic liver disease. This study is aimed at understanding the genetic causes of this group of conditions in children. Better understanding of the causes of cholestatic liver disease will improve diagnostic techniques, and is the first step toward improving treatment.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZDK1)
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Karp, Robert W
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University of California San Francisco
Internal Medicine/Medicine
Schools of Medicine
San Francisco
United States
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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