A custom-designed Next Generation Sequencing Panel for analysis of patients with assumed genetically determined cholestatic liver disease

Over the last decades, Sanger sequencing was the method of choice for the identification of genetic variants. Nowadays, advanced sequencing techniques, e.g. next generation sequencing (NGS), are in use to achieve a higher throughput. Genetic testing represents an important tool not only to confirm diagnosis but also for an individual therapeutic concept and risk stratification. We created a custom-designed NGS panel comprising the coding sequences and flanking intronic regions of 32 genes and moreover 16 specific hot spot regions. The panel includes well-known genes related to mono- and polygenetic cholestatic liver diseases as well as possible candidate genes involved in hepatobiliary transport and thus cholestasis. The hot spots cover specific genetic variants, which may promote progression of liver diseases (e.g. PNPLA3 p.I148 M, TM6SF2 p.E167K, HFE p.C282Y).

Genomic DNA of more than 90 patients with either a cholestatic phenotype or cryptogenic liver disease was analyzed using the customized NGS panel. About 120 different variants were detected in both patients and controls in the analyzed region, which spans about 126 kb.

To date 6 genes have been linked to progressive familial intrahepatic cholestasis. Besides the well-known genes encoding the hepatobiliary transport proteins FIC1, BSEP, and MDR3, which are associated with PFIC1 to PFIC3, the genes encoding TJP2, MYO5B, and FXR have been linked to this severe cholestatic phenotype. Besides numerous common polymorphisms, 43 different potentially disease-causing variants were identified in FIC1, BSEP, MDR3, TJP2, MYO5B, and FXR, respectively, including 6 novel variants. The DCDC2 gene has recently been linked high gamma GT cholestasis and has also been added to our NGS panel. Nevertheless, no potentially disease-causing variants, but 4 polymorphisms were identified in the DCDC2 gene so far. In addition to the confirmation of genetic-based cholestasis in several cases, the diagnosis of Dubin-Johnson syndrome was made for example in two succeeding generations of a family caused by a rare MRP2 missense mutation. Moreover, our NGS data underscores the hypothesis that multiple heterozygous variants in two or more genes may also cause a cholestatic phenotype.

In conclusion, we created a custom-designed NGS panel to evaluate the genetic background for patients with cholestatic disorders. It represents a fast and cost-effective tool to analyze a broad spectrum of genes simultaneously and is a valuable method for diagnostics and research. Nevertheless, the high amount of data obtained is a challenge and needs to be reviewed carefully using data bases, literature or in silico prediction tools.