The bile acid synthetic gene 3β-hydroxy-Δ5-C27-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis
Margrit Schwarz, Angelique C. Wright, Daphne L. Davis, Hisham Nazer, Ingemar Björkhem, David W. Russell
Margrit Schwarz, Angelique C. Wright, Daphne L. Davis, Hisham Nazer, Ingemar Björkhem, David W. Russell
View: Text | PDF
Article

The bile acid synthetic gene 3β-hydroxy-Δ5-C27-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis

Abstract

We used expression cloning to isolate cDNAs encoding a microsomal 3β-hydroxy-Δ5-C27-steroid oxidoreductase (C27 3β-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C19 and C21 3β-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C27 3β-HSD cDNA encodes an enzyme that is active against four 7α-hydroxylated sterols, indicating that a single C27 3β-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C19/21 steroids as substrates. The levels of hepatic C27 3β-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3β-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C27 3β-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.

Authors

Margrit Schwarz, Angelique C. Wright, Daphne L. Davis, Hisham Nazer, Ingemar Björkhem, David W. Russell

×

Figure 2

Options: View larger image (or click on image) Download as PowerPoint
Expression cloning of mouse cDNAs encoding C27 3β-HSD enzyme activity. C...
Expression cloning of mouse cDNAs encoding C27 3β-HSD enzyme activity. Cultured HEK 293 cells were cotransfected with a cDNA encoding the CYP7B1 oxysterol 7α-hydroxylase and pools of cDNAs derived from a mouse liver library. After 24 hours, the transfected cells were assayed for enzyme activity by thin-layer chromatography using [3H]cholest-5-ene-3β,25-diol (25-hydroxycholesterol, 1.2 μM) as substrate (see Methods). In lane 1, cells transfected with a control plasmid lacking a cDNA insert exhibited basal oxysterol 7α-hydroxylase activity, which converts 25-hydroxycholesterol into cholest-5-ene-3β,7α,25-triol, and low levels of C27 3β-HSD enzyme activity, which converts the product of the CYP7B1 enzyme into 7α,25-dihydroxy-cholest-4-ene-3-one. In lane 2, transfection with the CYP7B1 oxysterol 7α-hydroxylase cDNA alone increased the synthesis of cholest-5-ene-3β,7α,25-triol. In lane 3, transfection of a pool of 500 cDNAs containing a cDNA encoding C27 3β-HSD enzyme activity increased the synthesis of 7α,25-dihydroxy-cholest-4-ene-3-one. In lanes 4–6, transfection of progressively smaller (more enriched) pools containing the cDNA encoding the C27 3β-HSD activity resulted in a gradual increase in the synthesis of 7α,25-dihydroxy-cholest-4-ene-3-one and a decrease in the 7α-hydroxylated intermediate. In lanes 7 and 8 are radiolabeled sterol standards whose identities were confirmed previously by gas chromatography-mass spectrometry (12).