The power of NGS: Results from a dedicated sequencing panel of 24 genes in 40 patients with cholestatic liver disease

Background: Cholestasis indicates bile secretory failure, which might be caused by environmental factors and gene variants, or combination of both. Although there is variability in underlying causes of hepatocellular cholestasis, many patients develop cholestasis of unknown etiology and gene mutations of critical hepatobiliary transporters or their regulators may be suspected. Our aim now was to employ next generation sequencing (NGS) to dissect genetic risk factors in adult patients with cholestatic liver injury of unknown etiology.

Methods: The full length coding sequences of 24 genes known to be involved in regulation and transport of drugs and metabolites into bile (Table 1) were determined by NGS sequencing in selected patients with unexplained cholestatic liver injury, as defined by an at least two-fold increase of serum alkaline phosphatase (AP) activities and ALT/AP ratio ≤2. Population frequency and biochemical impact of all detected variants were analysed; rare, high-impact variants were also considered.

Tab. 1: Cholestasis sequencing panel:

ABCB4	ABCB11	ABCC2	ABCG5	ABCG8
ATP8B1	CIRH1A	CLDN1	JAG1	NOTCH2
NR1H4	NR1I2	UGT1A1 – 10	VIPAR	VPS33B

Results: Sequence analysis of 24 genes in 40 patients with cholestatic liver disease revealed heterozygosity for known disease-associated variants in 12 patients, and new high-impact potential candidate variants (splice site -1 or -2, frameshift deletions, rare non-conservative variants) in altogether 22 patients. Only 8/40 (20%) patients showed no credible variants.

Conclusions: A NGS panel of 24 cholestasis-related genes resulted in detection of creedal candidate gene variants in approximately three quarter of all index cases analysed. Only 4 of the 24 genes analysed have revealed no candidate variant to date. We are in the process of designing the next panel iteration by adding new candidate genes associated with cholestatic liver disease (CFTR, TJP2, GBPAR and others) and removing genes that have failed to turn up any candidate variants to date. Thus we are hoping to identify the underlying genetic cause of cholestasis in at least 90% of all cases analysed.

Corresponding author: Liebe, Roman

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