Next generation sequencing analysis of cholestatic patients reveals high impact variation in common and rare candidate genes: Deconstructing the genetic architecture of cholestasis

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) 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.
ABCB4 ABCB11 ABCC2 ABCG5 ABCG8
ATP8B1 CIRH1A CLDN1 JAG1 NOTCH2
NR1 H4 NR1I2 UGT1A1 – 10 VIPAR VPS33B
Results: Sequence analysis of 24 genes in 22 patients with cholestatic liver disease revealed heterozygosity for several high-impact variants that have previously been associated with cholestatic liver disease such as ABCB11 p.G982R (PFIC2), ABCB11 p.D482G (PFIC2), ATP8B1 p.I661T (PFIC1/BRIC), ATP8B1 p.D70N (BRIC/ICP) and VPS33B c.177+1G>A (ARC). New high-impact variants that are highly likely to result in non-functional protein (complete loss of splice site, frameshift deletion) were detected in ABCB4 (MDR3), NR1I2 (PXR) and UGT1A7. None of these variants were found among 6.500 individual sequences in the NHLBI exome database. Rare, non-conservative variants of yet unknown biochemical impact were also detected in ABCC2 (MRP2), NOTCH2 and NR1H4 (FXR).

Conclusions: Next generation sequencing permits rapid and comprehensive analysis of a large panel of cholestasis-associated transcripts. Peculiar phenotypes appear to be more likely to result in the detection of pathogenic or novel mutations. Correlating the results with systematic clinical phenotyping will result in improved genotype-phenotype correlations. Comprehensive genotyping might also permit targeted intervention if the respective mutations are amendable to chemical intervention such as chaperone-therapy.

Corresponding author: Liebe, Roman

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