Disclosures: The following people have nothing to disclose: Ammar Majeed, Annika Bergquist, Gunnar Söderdahl, Maria Castedal, Karouk Said Background: Next generation sequencing Roxadustat supplier (NGS) allows the high-throughput sequencing of multiple genes in several subjects, concomitantly. This new technology should lower the cost and time of molecular analyses in hereditary cholestasis and cholelithiasis, which comprise autosomal dominant and recessive disorders, and the disease-causing genes of which contain numerous exons. NGS might also help to

identify new genes in these disorders. Aims of the study : 1) Determine the feasibility and input of a NGS strategy for the screening of the genes implicated in the recessive disorders progressive familial intrahepatic cholestasis (i.e. ABCB4, ABCB11 and ATP8B1) and Dubin Johnson’s syndrome (i.e. ABCC2); 2) Screen 5 candidate genes encoding transporters (ABCG5, HM781-36B research buy ABCG8, SLC4A2) or bile acid receptors (NR1H4, GPBAR1). Materials

and Methods: Fifty-five consecutive unrelated patients (2/3 females), referred to our national reference laboratory for intra-hepatic cholestasis or cholelithiasis genotyping, were investigated. Genomic DNA was extracted from peripheral blood. A DNA capture strategy was developed, allowing the concomitant screening of 24 patients. Each DNA was converted to a sequencing library by fragmentation, end repair, and ligation

to oligonucleotide adapters. Specific gene probes for the 154 coding exons of the 10 genes of interest were designed with the Nimblegen software. Individual library fragments were double captured and clonally amplified by solid surface bridge amplification (MiSeq sequencer). The dropGenTM VAV2 application was used to analyze the raw data of sequencing. Results: NGS was extremely time-efficient and accurate. Each analysis of 24 patients was completed within a week. Sanger sequencing confirmed all identified variants. The mean coverage was ≥96.5% with a depth of 400X. In 37 patients, variants were detected in ABCB4 (n=14), ABCB11 (n=9), ABCC2 (n=14) and/or ATP8B1 (n=5) genes. In 5 patients, 2 of these genes were mutated (e.g. ATP8B1/ABCB4). In 10 patients, the variant(s) of one of these genes were associated with variant (s) in one of the candidate genes, namely GPBAR1 (n=4), ABCG5 (n=5) or ABCG8 (n=1). The homozygous ABCB11 p.Val444Ala genotype, known to be associated with hormonal cholestasis, was detected in 20 patients. Conclusions: NGS provides multiple advantages over the classical methods for genotyping subjects with hereditary cholestasis or cholelithiasis.