Sequencing of ATP8B1, ABCB11 and ABCB4 revealed 135 genetic variants in 374 unrelated patients with suspected intrahepatic cholestasis

ATP8B1, ABCB11, and ABCB4 encode the aminophospholipidflippase familial intrahepatic cholestasis 1 (FIC1), the bile salt export pump (BSEP), and the phospholipidfloppase multidrug resistance protein 3 (MDR3), respectively, that play central roles in bile formation. Mutations in these transporters are associated with cholestatic liver diseases of varying severity ranging from milder forms like intrahepatic cholestasis of pregnancy (ICP), benign recurrent intrahepatic cholestasis (BRIC) or low phospholipid-associated cholelithiasis (LPAC) to progressive familial intrahepatic cholestasis (PFIC). At present, gene sequencing is the main method to investigate the genetic background of these different types of intrahepatic cholestasis.

To confirm diagnosis based on the genetic background, 374 blood samples of unrelated patients presenting with a cholestatic phenotype of diverging manifestation were obtained from 14 different countries. Genomic DNA was used for sequencing analysis of all coding exons with surrounding intron regions of either ATP8B1, ABCB11 or ABCB4. For 81 patients, two or all three genes were sequenced.

27 variants including 6 new ones were detected in 119 samples of patients with assumed ATP8B1 (FIC1) deficiency. DNA sequencing from 187 patients with suspected ABCB11 (BSEP) mutations revealed 70 different variants, of which 36 represent novel variants. In 171 DNA samples, ABCB4 (MDR3) was analyzed which revealed 38 genetic variants comprising 18 novel ones. Nevertheless, in a variety of cases the genetic analysis uncovered only one heterozygous mutation for FIC1: 4/119 (3.4%), BSEP: 31/187 (16.6%), and MDR3: 27/171 (15.8%). Heterozygous mutations, common polymorphisms or synonymous variants probably not completely explain a severe cholestatic phenotype but should be considered as potential genetic factors for milder cholestatic diseases like BRIC, ICP or LPAC. In a number of patients without any grave mutation, common and/or synonymous variants were verifiable. The common FIC1 variants c.3531+8G>T and p.R952Q were detected in 39/105 (37.1%) samples. For BSEP, p.V444A combined with p.A1028A were demonstrated in 75/108 (69.4%) specimen. A combination of the three synonymous MDR3 variants p.L59L, p.N168N, and p.I237I was proven in 24/116 (20.7%) patients with no other mutation.

In the described patient population, 135 genetic variants were detected in ATP8B1, ABCB11 or ABCB4 including numerous cases with only one heterozygous or even no single mutation. This demonstrates that other genes such as the TJP2 which was recently described by Sambrotta and colleagues in cases of low GGT cholestasis, as well as non genomic factors contribute to the phenotype of some of these patients. In our study, we focused on the common and synonymous genetic variants in ATP8B1, ABCB11 or ABCB4 which seem to have a significant effect on the development of a cholestatic phenotype.

Corresponding author: Dröge, Carola

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