Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis

Abstract

Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.

Figure 1. Histological and immunohistochemical findings in 1HR1H4 mutant livers.

Expression of BSEP, MDR3 and FXR in liver (diaminobenzidine chromogen and hematoxylin counterstain) in patients with NR1H4-related cholestasis; normal controls in insets. Hematoxylin/eosin views for orientation. Original magnification, all images, × 200. BSEP and FXR are absent in all four patients when compared with positive control (inset). MDR3 is present in all four patients although reduced in patients 2 and 4. H&E, hematoxylin and eosin.

Figure 2. NR1H4 mutations.

(a) Sanger sequencing of the homozygous variant c.526C>T (p.R176*) in patients 1 and 2 of family 1. Both parents are heterozygous carriers. (b) Sequence of the homozygous variant c.419_420insAAA (p.Tyr139_Asn140insLys) in patients 3 and 4 of family 2. The mother carries a heterozygous change. (c) Breakpoint junction mapping in family 2. PCR and Sanger sequencing confirmed a 31.7 kb deletion that spans the first two coding exons of all NR1H4 isoforms. The deletion region is marked with a filled box. (d) All family 2 members except the mother carry this deletion. PCR was performed using forward and reverse primers indicated with arrow heads in d. Primer pair F1/R1b fails to amplify at the wild-type locus because the distance between them exceeds the limit of PCR reaction. Primer pair F1/R1 serves as control. NTC, no template control.

Figure 3. FXR Lys insertion mutation abrogates consensus IR-1 FXR binding and transactivation.

(a) The highly conserved zinc-finger domain of the human FXR protein. The residue of nonsense mutation found in family 1 is indicated with a green arrow. The location of the in-frame Lys 140 insertion found in family 2 is indicated with a red arrow. ‘Zn' stands for zinc ions. Cysteine residues that directly interact with zinc ions are in red. (b) Comparison of predicted structures of DBD. Predicted structures of wild-type FXR and FXR mutant with in-frame insertion based on the structure of EcR/RXR DBD in complex with IR-1 (PBD 1R0N). RXR is rendered in grey, EcR in orange, wild-type hFXR in cyan, hFXR mutant in purple and zinc ions are in green. The inserted lysine residue is in red with side chain shown. (c) Electrophoretic mobility-shift assay performed with a consensus FXRE or the FXR-binding region in mouse SHP promoter and wild-type or Lys insertion mutant FXR and RXR as indicated. (d) Relative luciferase expression directed by a reporter containing three copies of a consensus in response to wild-type and mutant FXR proteins in the presence or absence of the synthetic FXR agonist GW4064. Repeated three times (*P, 0.01 by t-test, error bars s.d.). FXRE, FXR-binding element.