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Characterization of disease-related 5beta-reductase (AKR1D1) mutations reveals their potential to cause bile acid deficiency.

ABSTRACT: Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5beta-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5beta-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Delta(4) double bond in 3-keto steroids and sterols to yield the 5beta-hydrogenated product. Analysis of the AKR1D1 gene in five patients with 5beta-reductase deficiency revealed five different mutations resulting in an amino acid substitution in the protein. To investigate a causal role for these observed point mutations in AKR1D1 in 5beta-reductase deficiency, we characterized their effect on enzymatic properties. Attempts to purify mutant enzymes by overexpression in Escherichia coli only yielded sufficient amounts of the P133R mutant for further characterization. This enzyme displayed a highly reduced K(m) and V(max) reminiscent of uncompetitive kinetics with 4-cholesten-7alpha-ol-3-one as substrate. In addition, this mutant displayed no change in cofactor affinity but was more thermolabile in the absence of NADPH as judged by CD spectroscopy. All mutants were compared following expression in HEK 293 cells. Although these enzymes were equally expressed based on mRNA levels, protein expression and functional activity were dramatically reduced. Cycloheximide treatment also revealed that several of the expressed mutants were less stable. Our findings show that the reported mutations in AKR1D1 in patients with 5beta-reductase lead to significantly decreased levels of active enzyme and could be causal in the development of bile acid deficiency syndrome.

SUBMITTER: Drury JE

PROVIDER: S-EPMC2915689 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Characterization of disease-related 5beta-reductase (AKR1D1) mutations reveals their potential to cause bile acid deficiency.

Drury Jason E JE Mindnich Rebekka R Penning Trevor M TM

The Journal of biological chemistry 20100603 32

Bile acid deficiency is a serious syndrome in newborns that can result in death if untreated. 5beta-Reductase deficiency is one form of bile acid deficiency and is characterized by dramatically decreased levels of physiologically active 5beta-reduced bile acids. AKR1D1 (aldo-keto reductase 1D1) is the only known human enzyme that stereo-specifically reduces the Delta(4) double bond in 3-keto steroids and sterols to yield the 5beta-hydrogenated product. Analysis of the AKR1D1 gene in five patient ...[more]

PMID: 20522910

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Project description:BackgroundA substantial group of patients with cholestatic liver disease in infancy excrete, as the major urinary bile acids, the glycine and taurine conjugates of 7alpha-hydroxy-3-oxo-4-cholenoic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholenoic acid. It has been proposed that some (but not all) of these have mutations in the gene encoding delta(4)-3-oxosteroid 5beta-reductase (SRD5B1; AKR1D1, OMIM 604741).AimsOur aim was to identify mutations in the SRD5B1 gene in patients in whom chenodeoxycholic acid and cholic acid were absent or present at low concentrations in plasma and urine, as these seemed strong candidates for genetic 5beta-reductase deficiency.Patients and subjectsWe studied three patients with neonatal onset cholestatic liver disease and normal gamma-glutamyl transpeptidase in whom 3-oxo-delta(4) bile acids were the major bile acids in urine and plasma and saturated bile acids were at low concentration or undetectable. Any base changes detected in SRD5B1 were sought in the parents and siblings and in 50 ethnically matched control subjects.MethodsDNA was extracted from blood and the nine exons of SRD5B1 were amplified and sequenced. Restriction enzymes were used to screen the DNA of parents, siblings, and controls.ResultsMutations in the SRD5B1 gene were identified in all three children. Patient MS was homozygous for a missense mutation (662 C>T) causing a Pro198Leu amino acid substitution; patient BH was homozygous for a single base deletion (511 delT) causing a frame shift and a premature stop codon in exon 5; and patient RM was homozygous for a missense mutation (385 C>T) causing a Leu106Phe amino acid substitution. All had liver biopsies showing a giant cell hepatitis; in two, prominent extramedullary haemopoiesis was noted. MS was cured by treatment with chenodeoxycholic acid and cholic acid; BH showed initial improvement but then deteriorated and required liver transplantation; RM had advanced liver disease when treatment was started and also progressed to liver failure.ConclusionsAnalysis of blood samples for SRD5B1 mutations can be used to diagnose genetic 5beta-reductase deficiency and distinguish these patients from those who have another cause of 3-oxo-delta(4) bile aciduria, for example, severe liver damage. Patients with genetic 5beta-reductase deficiency may respond well to treatment with chenodeoxycholic acid and cholic acid if liver disease is not too advanced.

Project description:BACKGROUND:Disorders of primary bile acid synthesis may be life-threatening if undiagnosed, or not treated with primary bile acid replacement therapy. To date, there are few reports on the management and follow-up of patients with ?4-3-oxosteroid 5?-reductase (AKR1D1) deficiency. We hypothesized that a retrospective analysis of the responses to oral bile acid replacement therapy with chenodeoxycholic acid (CDCA) in patients with this bile acid synthesis disorder will increase our understanding of the disease progression and permit evaluation of this treatment regimen as an alternative to the Food and Drug Administration (FDA) approved drug cholic acid, which is currently unavailable in China. AIM:To evaluate the therapeutic responses of patients with AKR1D1 deficiency to oral bile acid therapy, specifically CDCA. METHODS:Twelve patients with AKR1D1 deficiency, confirmed by fast atom bombardment ionization-mass spectrometry analysis of urine and by gene sequencing for mutations in AKR1D1, were treated with differing doses of CDCA or ursodeoxycholic acid (UDCA). The clinical and biochemical responses to therapy were monitored over a period ranging 0.5-6.4 years. Dose adjustment, to optimize the therapeutic dose, was based on changes in serum biochemistry parameters, notably liver function tests, and suppression of the urinary levels of atypical hepatotoxic 3-oxo-?4-bile acids measured by mass spectrometry. RESULTS:Physical examination, serum biochemistry parameters, and sonographic findings improved in all 12 patients during bile acid therapy, except one who underwent liver transplantation. Urine bile acid analysis confirmed a significant reduction in atypical hepatotoxic 3-oxo-?4 bile acids concomitant with clinical and biochemical improvements in those patients treated with CDCA. UDCA was ineffective in down-regulating endogenous bile acid synthesis as evidenced from the inability to suppress the urinary excretion of atypical 3-oxo-?4-bile acids. The dose of CDCA required for optimal clinical and biochemical responses varied from 5.5-10 mg/kg per day among patients based on maximum suppression of the atypical bile acids and improvement in serum biochemistry parameters, and careful titration of the dose was necessary to avoid side effects from CDCA. CONCLUSION:The primary bile acid CDCA is effective in treating AKR1D1 deficiency but the therapeutic dose requires individualized optimization. UDCA is not recommended for long-term management.

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Characterization of disease-related 5beta-reductase (AKR1D1) mutations reveals their potential to cause bile acid deficiency.

Publications

Characterization of disease-related 5beta-reductase (AKR1D1) mutations reveals their potential to cause bile acid deficiency.

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