A novel strategy for the large scale identification of unknown PCLD genes


General background
Autosomal dominant polycystic liver disease (PCLD) presents with multiple liver cysts that cause symptoms as the liver grows to several times its original volume and displaces surrounding abdominal organs. Genetic linkage studies in large families have identified 2 genes, PRKCSH and SEC63, that can cause PCLD. There is considerable genetic heterogeneity and only 25% of PCLD patients have a PRKCSH or SEC63 mutation. We recently discovered that 70% of heterozygous PRKCSH mutation carriers had lost the other functioning PRKCSH allele. This loss of heterozygosity specifically occurs in cyst epithelium.

Objective
A novel strategy to identify unknown genes in polycystic liver disease.

Hypothesis
We hypothesize that somatic loss of heterozygosity is a general principle that underlies PCLD and identification of this second hit in a PCLD patient will point us to the first hit, i.e. a mutation in a novel PCLD gene.

Aim
We want to create a roadmap with genomic regions displaying loss of heterozygosity in cyst epithelia of the 75% patients without a known gene mutation. We will use this information to identify 5- 10 novel PCLD genes and thereby significantly increase the diagnostic yield in this patient group. Methods: Our proposal involves 3 sequential steps (1) Isolation of liver cyst epithelium using laser dissection microscopy of available and prospectively collected tissues & isolation of cyst epithelial cells from liver cyst fluid using cell sorting. (2) DNA isolation from cyst epithelium followed by a genome wide SNP analysis, which will identify regions with loss of heterozygosity. (3) Gene discovery using a dual genomic partitioning strategy based upon a candidate gene approach and next generation sequencing.

Anticipated results
We expect that our novel strategy directly results in: 1. Direct and large scale isolation of DNA from PCLD patient cyst epithelium 2. Identification of genomic regions with somatic loss of heterozygosity 3. Discovery of new PCLD genes

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