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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 4 1833-1841
Copyright © 2003 by The Endocrine Society

Molecular Genetics of 3ß-Hydroxy-{Delta}5-C27-Steroid Oxidoreductase Deficiency in 16 Patients with Loss of Bile Acid Synthesis and Liver Disease

Jeffrey B. Cheng, Emmanuel Jacquemin, Marie Gerhardt, Hisham Nazer, Danièle Cresteil, James E. Heubi, Kenneth D. R. Setchell and David W. Russell

Department of Molecular Genetics (J.B.C., D.W.R.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046; Department of Biochemistry (M.G.), St. Joseph Hospital, 75674 Paris, France; Pediatric Hepatology and Inserm U347 (E.J., D.C.), Bicêtre University Hospital and Public Assistance Hospital of Paris, 94275 Paris, France; Department of Pediatrics (H.N.), King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 11211; and Department of Pediatrics (J.E.H., K.D.R.S.), Cincinnati Children’s Hospital, Cincinnati, Ohio 45229

Address all correspondence and requests for reprints to: Dr. David W. Russell, Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9046. E-mail: david.russell{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The 3ß-hydroxy-{Delta}5-C27-steroid oxidoreductase (C27 3ß-HSD) is a membrane-bound enzyme of the endoplasmic reticulum that catalyzes an early step in the synthesis of bile acids from cholesterol. Subjects with autosomal recessive mutations in the encoding gene, HSD3B7, on chromosome 16p11.2–12 fail to synthesize bile acids and develop a form of progressive liver disease characterized by cholestatic jaundice and malabsorption of lipids and lipid-soluble vitamins from the gastrointestinal tract. The gene encoding the human C27 3ß-HSD enzyme was isolated previously, and a 2-bp deletion in exon 6 of HSD3B7 was identified in a well characterized subject with this disorder. Here, we report a molecular analysis of 15 additional patients from 13 kindreds with C27 3ß-HSD deficiency. Twelve different mutations were identified in the HSD3B7 gene on chromosome 16p11.2–12. Ten mutations were studied in detail and shown to cause complete loss of enzyme activity and, in two cases, alterations in the size or amount of the transcribed mRNA. Mutations were inherited in homozygous form in 13 subjects from 10 families and compound heterozygous form in four subjects from three families. We conclude that a diverse spectrum of mutations in the HSD3B7 gene underlies this rare form of neonatal cholestasis.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CONVERSION OF cholesterol into bile acids is essential for the maintenance of cholesterol homeostasis, regulation of hepatic function, and absorption of fats and fat-soluble vitamins from the gastrointestinal tract. Inherited mutations that decrease the synthesis of bile acids or their transport cause a disorder beginning in infants and consisting of impairment of liver function because of decreased bile flow (cholestasis) and lipid and vitamin malabsorption.

The genetic basis of neonatal cholestasis is heterogeneous. Mutations in three genes encoding primary bile acid biosynthetic enzymes have so far been identified as causes of this disease. These include the oxysterol 7{alpha}-hydroxylase gene (CYP7B1) (1) located on chromosome 8q21.3, the 3-oxo-{Delta}4-steroid 5ß-reductase gene (AKR1D1) (2) on chromosome 7q32–33, and the 3ß-hydroxy-{Delta}5-C27-steroid oxidoreductase (C27 3ß-HSD) gene (HSD3B7) (3, 4)) on chromosome 16p11.2–12. Each of the encoded enzymes catalyzes an early step in the biosynthetic pathway, and, consequently, their loss prevents the synthesis of adequate levels of primary bile acids required for the promotion of bile secretion and intraluminal fat absorption.

Deficiencies in bile acid transport causing neonatal cholestasis also have been traced to mutations in three genes, including FIC1 on chromosome 18q21 (5), ABCB11 on chromosome 2q24 (6), and ABCB4 on chromosome 7q21.1 (7). The product of FIC1 is a P-type ATPase expressed on the apical membranes of enterocytes in the small intestine and to a lesser extent in liver parenchyma. The ABCB11 gene product often is referred to as the bile salt export protein and is expressed on the hepatocyte canalicular membrane. The protein specified by the ABCB4 gene, originally designated the multidrug resistance 3 protein, is located on the canalicular membrane of the hepatocyte and is active in the transport of phospholipids. Both ABCB11 and ABCB4 are members of a large family of proteins that transport small molecules out of cells (8, 9).

Although neonatal cholestasis is genetically heterogeneous, the manifestations of loss of either a biosynthetic enzyme or transport protein are similar. Affected individuals present at birth or in early childhood with cholestatic jaundice, fat-soluble vitamin deficiency, and acholic or fatty stools (steatorrhea). Serum transaminases are usually elevated, and a conjugated hyperbilirubinemia is often present. Liver biopsies may reveal nonspecific changes, but giant cell transformation of hepatocytes, inflammation, fibrosis, and canalicular and hepatocyte cholestasis is usual (10, 11, 12). Chemical analysis of body fluids reveals an accumulation of atypical bile acids and sterol intermediates. Bile acid biosynthetic defects, in particular C27 3ß-HSD deficiency, may also cause late-onset chronic cholestasis, and in these individuals the clinical history generally reveals a pattern of mildly elevated transaminases in infancy that often resolves only to reemerge later and an early-onset of vitamin D-deficient rickets (13).

Despite a shared clinical presentation, the various genetic forms of neonatal cholestasis require different treatments. Loss of either the C27 3ß-HSD or the 3-oxo-{Delta}4-steroid 5ß-reductase enzyme is treated by oral administration of bile acids (11, 14, 15, 16), a therapy that is both effective and relatively free of side effects. In contrast, loss of the oxysterol 7{alpha}-hydroxylase enzyme (1) or various bile acid transporters (17) requires liver transplantation. The marked differences in the treatment regimens for the various forms of inherited neonatal cholestasis underscore the importance of defining the molecular basis of the disease in individual patients (18).

In this study, we describe the molecular basis of neonatal cholestasis caused by C27 3ß-HSD deficiency in a cohort of patients diagnosed by mass spectrometric analyses in Paris and Cincinnati (19). Affected individuals in this cohort are either homozygous or compound heterozygous carriers of 12 different mutations in the HSD3B7 gene, many of which are shown to inactivate the encoded enzyme. Notwithstanding this molecular heterogeneity, diagnosis of the disease and subsequent treatment with bile acids led to favorable outcomes in most patients.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Institutional Review Board approvals were obtained from the University of Texas Southwestern Medical Center, Bicêtre University Hospital, King Faisal Specialist Hospital, St. Joseph Hospital, and the Cincinnati Children’s Hospital. Informed consent was obtained from all subjects, and consent forms are maintained at the above institutions. Oral bile acid therapy with cholic acid was provided as an investigational new drug approved by the Food and Drug Administration. A preliminary medical history of probands from families A–E has been reported (19). Salient clinical features of these and other affected individuals in the cohort analyzed here are summarized in Table 1Go.


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  		Table 1. Summary of clinical data from 16 patients with C27 3ß-HSD deficiency

 
Materials and methods

Genomic DNA extraction and sequencing. Human genomic DNA was isolated from white blood cells or cultured fibroblasts using Puregene DNA isolation kits (Gentra Systems, Minneapolis, MN). Exons of the HSD3B7 gene were amplified by PCR using the Advantage-GC genomic PCR kit (CLONTECH Laboratories, Inc., Palo Alto, CA). DNA products were purified by centrifugation through Centricon YM-100 filter devices (Millipore Corp., Billerica, MA) and subjected to sequence analysis using a thermostable DNA polymerase and fluorescently labeled nucleoside terminators (Applied Biosystems, Foster City, CA). Data were collected on an automated DNA sequencer (Applied Biosystems).

Biochemical analysis of HSD3B7 alleles. A fragment of DNA corresponding to nucleotides 181,011 through 178,216 of the Homo sapiens chromosome 16 working draft sequence (accession no. NT_024826.3) that encompassed exons 1 through 6 of the HSD3B7 gene was amplified from each proband’s genomic DNA. The primers were designed to introduce an SalI site at the 5'-end of the amplified DNA and an NotI site at the 3'-end. Amplification reactions contained 2.5 U Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and 5% (vol/vol) dimethylsulfoxide. The resulting DNA fragment was ligated into the pCMV6 expression vector (GenBank accession no. 239250), and the presence of the appropriate mutation was confirmed by DNA sequence analysis. A small fragment of DNA containing each mutation was then substituted for the corresponding segment of the normal gene harbored in pCMV6.

Human embryonic kidney (HEK) 293 cells (CRL 1573; American Type Culture Collection, Manassas, VA) were plated at a density of 4 x 105 cells per 60-mm dish in 2.5 ml DMEM containing 1 g/liter glucose, 10% (vol/vol) fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Two days after plating, cells were transfected using the FuGENE6 reagent (Roche Diagnostics, Indianapolis, IN) with a mixture of plasmid DNAs. The mixture included 1.8 µg pCMV6-hCYP7B1, encoding the human oxysterol 7{alpha}-hydroxylase enzyme (1), 1.8 µg pCMV6-HSD3B7 (mutant or normal), and 0.4 µg pVA-1, a vector expressing the adenovirus type 5 VA1 gene (20). At 16 h post transfection, the FuGENE6-containing medium was replaced with fresh DMEM medium supplemented with 0.0152 µM [3H]cholest-5-ene-3ß,25-diol (25-hydroxycholesterol, 78.5 Ci/mmol, NEN Life Science Products, Boston, MA), and 1.0 µM 25-hydroxycholesterol. The medium was harvested 8 h later, and lipids were extracted using 8 ml Folch reagent (chloroform:methanol; 2:1, vol/vol) per dish. The organic extract was lyophilized and lipids were dissolved in 50 µl acetone and resolved by thin-layer chromatography on pre-cored LK5DF silica gel plates (Whatman, Clifton, NJ) in a solvent system of toluene:ethyl acetate (2:3, vol/vol). Radioactive sterols were detected by phosphor imaging on a BAS1000 machine (Fuji Photo Film Co., Ltd. Medical Systems, Tokyo, Japan) or autoradiography using X-OMAT AR film (Kodak, Rochester, NY). The detection limit for this enzymatic assay was 0.05–0.2 pmol C27 3ß-HSD product/min per milligram cell protein.

Analysis of HSD3B7 RNA. HEK 293 cells were transfected as described above with pCMV6 constructs harboring either a normal C27 3ß-HSD gene fragment or a variant allele. At 24 h after transfection, total cellular RNA was isolated using RNA STAT-60 (Tel-Test "B" Inc.) and poly(A)+-enriched mRNA was isolated by oligo(dT)-cellulose chromatography (mRNA purification kit, Amersham Pharmacia Biotech, Piscataway, NJ). Aliquots of purified mRNA (5 µg) were separated by electrophoresis through 1.4% (wt/vol) agarose gels and subjected to blot hybridization using standard procedures (21). Radiolabeled probes were derived from a human C27 3ß-HSD cDNA (GenBank accession no. AF277719; Ref. 4) and ß-actin cDNA (GenBank accession no. NM_001101;22).

Database access. Accession numbers and URLs for data in this article are: GenBank, http://www.ncbi.nih.gov/GenBank/[for human CYP7B1 cDNA (accession no. AF029403), pCMV6 (accession no. AF029403), C27 3ß-HSD cDNA (accession no. AF277719), human C27 3ß-HSD gene (accession no. NT_024826.3), human ß-actin (accession no. NM_001101)]; Online Mendelian Inheritance of Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/[for giant cell hepatitis, neonatal (MIM231100), oxysterol 7{alpha}-hydroxylase 1, CYP7B1 (MIM603711), 3-oxo-{Delta}4-steroid 5ß-reductase (MIM235555), FIC1 (MIM602397), ABCB11 (MIM603201), MDR3 (MIM602347)].


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
HSD3B7 mutations in patients with neonatal cholestasis

The diagnosis of C27 3ß-HSD deficiency in probands was based on clinical presentation and mass spectrometric identification in urine of the signature 3ß-hydroxy-{Delta}5 bile acids characteristic of the biochemical defect (Fig. 1Go). To confirm the diagnosis and establish the molecular basis of the disorder, genomic DNA was isolated from the white blood cells of probands and, when possible, other affected and unaffected family members. The data of Fig. 2Go, A and B, show two extended Arabic pedigrees in which C27 3ß-HSD deficiency segregated as an apparent autosomal recessive trait. This inheritance pattern was confirmed in family J by DNA sequence analysis of exon 6 in which a 2-bp deletion (1057, {Delta}CT) was detected in heterozygous form in the parents and unaffected carrier offspring and in homozygous form in the proband and an affected sibling (Fig. 2BGo).



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  		Figure 1. Reaction catalyzed by the C27 3ß-HSD enzyme. A generic 7{alpha}-hydroxylated intermediate in the bile acid biosynthetic pathway is shown at left. The C27 3ß-HSD reaction involves isomerization of the {Delta}5 bond to {Delta}4 bond and the oxidation of the 3ß-hydroxyl group to an oxo group. The actions of an additional 9 or 10 enzymes produce primary bile acids. Mutations in the C27 3ß-HSD enzyme cause the accumulation in the plasma and urine of 7{alpha}-hydroxylated intermediates with 3ß-hydroxyl, {Delta}5 structures.

 


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  		Figure 2. Pedigrees of two families with C27 3ß-HSD deficiency. The two kindreds are designated by a capital letter (I and J) with generations designated by a Roman numeral and each individual within a generation by an Arabic number. An arrow designates the proband in each family. Question marks designate individuals of unknown HSD3B7 genotype. The DNA sequence of a small segment of exon 6 from the HSD3B7 genes of seven family J individuals is shown below the pedigree in B. The 1057, {Delta}CT mutation segregates in an autosomal recessive manner in this family.

 
Amplifying a 3-kb segment that encompassed the six exons and five introns of the gene and subjecting this DNA to sequence analysis identified additional mutations in HSD3B7. These experiments revealed 12 different mutations segregating in the 13 kindreds analyzed (Fig. 3Go). In 10 of the 12 families, including all of those with confirmed or suspected consanguinity, a single mutation was found in homozygous form. The probands in the remaining three kindreds were compound heterozygotes who inherited two different mutations in the HSD3B7 gene. The molecular genetic features of this cohort are summarized in Table 2Go.



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  		Figure 3. DNA sequence analyses of mutations in the HSD3B7 gene. The sequences of small segments of the gene are shown together with the locations of 10 mutations identified in 12 families.

 

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  		Table 2. Molecular genetics of C27 3ß-HSD deficiency

 
Individual mutations mapped to four exons and two introns of HSD3B7 and included one nonsense mutation, two missense mutations, five small deletions of one to three base pairs, two small insertions of one to two base pairs, and two mutations that altered consensus splice junction sequences (Fig. 4Go).



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  		Figure 4. Structure of the HSD3B7 gene and locations of mutations identified in subjects with neonatal cholestasis. The structure of the gene is drawn to scale with exons indicated by boxes and introns by interconnecting lines. Amino acids occurring at exon/intron boundaries are indicated in single letter code above the gene schematic. The locations of 12 mutations identified in 16 patients are shown below the schematic together with the chromosomal assignment of the HSD3B7 gene. The nucleotide numbers indicating the positions of individual mutations refer to those of the human C27 3ß-HSD cDNA (GenBank accession no. AF277719).

 
Analysis of HSD3B7 mRNA

The effects of 10 of these mutations on the size and amount of mRNA transcribed from the mutant HSD3B7 alleles were determined. Individual mutations were recreated by site-directed mutagenesis in a pCMV expression vector containing a 3-kb segment of genomic DNA encompassing the gene. Transcription from these minigene constructs is initiated within a cytomegalovirus promoter and terminated within sequences of the human GH gene, and splicing signals are supplied by the introns of the HSD3B7 gene. Individual plasmid DNAs were introduced into HEK 293 cells by transfection, and levels of mature C27 3ß-HSD mRNA were determined by blot hybridization.

The data of Fig. 5Go show that cells transfected with the pCMV vector alone do not produce a C27 3ß-HSD mRNA (lane 1), whereas those transfected with a construct containing the normal gene synthesize an mRNA that is approximately 1.5 kb in length (lane 2). All but two of the eight mutant genes analyzed in this experiment produced a similarly sized mRNA in approximately the same amount. The two exceptions were genes containing the 132, iCC mutation in exon 1 (lane 5), and the 713–1, G->A mutation in the splice acceptor site of intron 5 (lane 9). Little or no mature C27 3ß-HSD mRNA was detected in cells transfected with the 132, iCC mutation, presumably because this lesion leads to rapid turnover of the transcribed mRNA. The 713–1, G->A mutation gave rise to a small amount of normal-sized mRNA and a larger RNA of approximately 2.7 kb. Although not tested directly, the size of the larger RNA suggested that it might contain sequences corresponding to the unspliced intron 5 in addition to those derived from exons 1 through 5. Constructs containing two additional HSD3B7 mutations, 63, {Delta}AG and 1057, {Delta}CT, were analyzed in a separate experiment and found to produce mRNAs that were similar in size and amount to those derived from the normal gene (data not shown). The effects of the G19S and 140, {Delta}TC mutations on steady-state mRNA levels were not determined.



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  		Figure 5. RNA blotting analysis of HSD3B7 mutations. A segment of genomic DNA spanning exons 1–6 of the C27 3ß-HSD gene and containing either the normal sequence or indicated mutation was ligated into the pCMV6 expression vector. The resulting plasmids were introduced into cultured HEK 293 cells by transfection. After a 16-h period of expression, poly(A)+ RNA was isolated from the transfected cells and analyzed by blot hybridization with a radiolabeled probe derived from the C27 3ß-HSD cDNA (top). The filter was stripped of radioactivity and hybridized again with a radiolabeled probe derived from a ß-actin cDNA (bottom). The positions to which RNAs of known size migrated in the gel are indicated on the left of the autoradiograms.

 
Analysis of enzyme activity

The C27 3ß-HSD enzyme catalyzes the isomerization and oxidation of 7{alpha}-hydroxylated sterol intermediates that arise in the pathways of bile acid synthesis. These substrates are neither commercially available nor readily synthesized in radiolabeled form. To overcome these challenges, an assay was employed in which 7{alpha}-hydroxylated substrates are produced in situ by incubating cells transfected with cDNAs encoding sterol 7{alpha}-hydroxylase enzymes with radiolabeled sterol precursors (4). For example, incubation of HEK 293 cells transfected with the CYP7B1 oxysterol 7{alpha}-hydroxylase cDNA with 25-hydroxycholesterol, resulted in the production of [3H]cholest-5-ene-3ß,7{alpha},25-triol (Fig. 6Go, lane 2), a 7{alpha}-hydroxylated substrate of the C27 3ß-HSD enzyme. Monitoring the conversion of this sterol into 7{alpha},25-dihydroxycholest-4-ene-3-one, in cells cotransfected with cDNA expression vectors encoding both CYP7B1 and C27 3ß-HSD, allowed measurement of C27 3ß-HSD enzyme activity (lane 3).



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  		Figure 6. Effects of HSD3B7 mutations on C27 3ß-HSD enzyme activity. Expression vectors harboring genomic DNAs bearing the indicated normal or mutant alleles of the HSD3B7 gene were introduced into cultured HEK 293 cells by transfection. Subsequent enzyme activity measurements in whole cells were performed as described in Subjects and Methods. A phosphor image arising from a scan of a thin-layer chromatography plate is shown. The positions to which sterols of known structure migrated on the plate are shown on the right of the image. Only cells transfected with the normal HSD3B7 gene (lane 3) expressed an enzyme capable of converting cholest-5-ene-3ß,7{alpha},25-triol into 7{alpha},25-dihydroxy-cholest-4-ene-3-one. Approximately five times more of the starting substrate (cholest-5-ene-3ß,25-diol) was chromatographed in lane 1 vs. the other lanes.

 
When expression plasmids containing eight of the mutant HSD3B7 genes were transfected into HEK 293 cells together with the CYP7B1 cDNA, little or no conversion of [3H]cholest-5-ene-3ß,7{alpha},25-triol into the C27 3ß-HSD product was observed (Fig. 6Go, lanes 4–11). In separate experiments, minigenes harboring the 63, {Delta}AG and 1057, {Delta}CT mutations also failed to encode an active enzyme when introduced into HEK 293 cells (data not shown). These results indicated that 10 of the 12 mutations identified in the HSD3B7 gene were null alleles, at least as judged at the level of sensitivity (0.05–0.2 pmol C27 3ß-HSD product/min per milligram cell protein) of the biochemical assay employed. The effects of the two remaining mutations (G19S and 140, {Delta}TC) were not determined. Antibodies directed against the human C27 3ß-HSD were not available; thus, we were unable to determine which of the mutant alleles directed the synthesis of a stable protein.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We describe a genetic and molecular analysis in 16 patients from 13 families with a form of neonatal cholestasis arising from C27 3ß-HSD deficiency. A diverse spectrum of point mutations, small insertions, and deletions in the HSD3B7 genes of affected individuals are present in homozygous and compound heterozygous form. These lesions variably affect the steady-state levels of mRNA transcribed from the mutant genes and in every case tested impair synthesis of an active C27 3ß-HSD enzyme. In turn, this loss prevents the synthesis of bile acids and disrupts liver function.

It has been recognized since 1987 that this disorder is the result of a defect in an early step of bile acid synthesis (3). We previously showed that the molecular basis of this genetic disease in one well-studied patient from Saudi Arabia was due to a mutation in the HSD3B7 gene (4). The present study reveals that C27 3ß-HSD deficiency is molecularly heterogeneous but genetically and therapeutically homogeneous. Molecular heterogeneity is indicated by the identification of 12 different mutations in the gene. The existence of so many different mutations does not complicate the molecular diagnosis of the disease because the HSD3B7 gene spans only 3 kb of DNA and is thus readily amplified and sequenced in its entirety. Genetic homogeneity is evident from the findings that all patients presenting with documented or suspected (see below) urinary accumulations of 3ß,7{alpha}-dihydroxy-5-cholenoic acid and 3ß,7{alpha},12{alpha}-trihydroxy-5-cholenoic acid inherited mutations in the HSD3B7 gene. Thus, unlike other bile acid biosynthetic enzymes, which exist in multiple isoforms (23), there appears to be only one human gene that encodes C27 3ß-HSD enzyme activity. A positive response to oral bile acids in all but two of the patients harboring different mutations shows the therapeutic homogeneity in C27 3ß-HSD deficiency (Table 1Go).

Mutations in HSD3B7 are rare and account for only a small percentage of neonatal cholestasis. In agreement with this low frequency, the probands from 10 of the 12 families studied here are true homozygotes who inherited identical mutations from both parents (Table 2Go). The remaining three probands are compound heterozygotes. The 310, iC, E147K, 1057, {Delta}CT, and 63, {Delta}AG mutations were each present in two families, although there were no known shared histories between family pairs with the same mutation. This outcome suggests that these four mutations have been present for extended periods of time in the population, presumably existing in heterozygous form, because before the advent of bile acid therapy in the 20th century, individuals inheriting two mutations in the gene would have almost certainly perished before sexual maturation.

The OMIM classifies C27 3ß-HSD deficiency under the general heading of "giant cell hepatitis, neonatal" (OMIM 231100), together with various forms of neonatal hemochromatosis caused by mutations in the steroid 5ß-reductase (AKR1D1) and oxysterol 7{alpha}-hydroxylase (CYP7B1) genes of bile acid biosynthesis. The detailed clinical descriptions published in the literature together with the isolation of the encoding gene and the molecular analyses presented in this study and elsewhere (4) clearly indicate that C27 3ß-HSD deficiency is a distinct disease entity.

The mutations identified here shed little light on the biochemistry of C27 3ß-HSD, a 369-amino acid enzyme that is located in the endoplasmic reticulum membrane and has an unknown topology (4, 24). Six of the 12 mutations map to the first two exons of the gene and are predicted to encode severely truncated proteins (Fig. 4Go). As expected, these fragments do not possess enzyme activity (Fig. 6Go). Similarly, the loss of one (1042, {Delta}T) or two (1057, {Delta}CT) base pairs from the last exon of the gene shifts the translational reading frame and eliminates enzyme activity. The protein encoded by the 1042, {Delta}T mutation is composed of 341 amino acids from the normal protein fused to a 74-residue extension at the C terminus, whereas the 1057, {Delta}CT mutation encodes a protein with the first 346 amino acids of the normal protein fused to a five-residue extension. In these cases, we do not know whether inactivation of the enzyme results from elimination of the normal C-terminal sequences or the addition of spurious amino acids at this end.

Only two potentially informative substitution mutations were identified in this survey. A G19S mutation in exon 1 was detected in the proband of family M while the manuscript was being reviewed and was not analyzed further. The proband of family F inherited a G-to-A transition mutation in exon 4 that results in the substitution of a lysine residue for glutamate at position 147 (E147K). Although an apparently stable mRNA of normal abundance was transcribed from the mutant gene (Fig. 5Go), no enzyme activity was detected in the transfected cells (Fig. 6Go). The C27 3ß-HSD is a member of a small family of enzymes that metabolize steroids and share sequence identity (4). In sequence alignments of the seven known family members, the glutamate 147 residue of C27 3ß-HSD is conserved in all of them, and this residue is shared also among the human, mouse, and rat C27 3ß-HSD enzymes (4). The preservation of this glutamate is suggestive of an important catalytic or structural role in these isomerase/dehydrogenases and may explain why substitution with lysine inactivates the C27 3ß-HSD enzyme in the proband. The glycine residue at position 19 also is conserved in all members of the 3ß-HSD enzyme family, and its loss may inactivate the C27 3ß-HSD in the patient from family M.

Because all of the mutations analyzed in detail here were null alleles, we were unable to deduce any phenotype-genotype correlations in this cohort of C27 3ß-HSD-deficient patients.

The availability of chemical and molecular methods to diagnose C27 3ß-HSD deficiency raises questions concerning the relative merits of each procedure and whether there are cases in which one or the other method, or both, are needed for an unambiguous assignment. Ascertainment by chemical procedures requires sophisticated mass spectrometry that is available only in a small number of medical centers. Diagnosis by molecular methods is more widely possible but still requires access to appropriate facilities and expertise. While this manuscript was in review, we encountered a case in which both methods were required to make an accurate diagnosis of C27 3ß-HSD deficiency. The mass spectra of samples obtained from the proband of family M were suggestive of the disorder but not definitive as a consequence of the advanced liver disease in this patient and the presence of obscuring ions arising from the ursodeoxycholic acid that was prescribed at the first signs of cholestasis. Analysis of the proband’s DNA, which revealed two mutations in exon 1 of the gene (G19S and 140, {Delta}TC), was required to make an unambiguous assignment of the deficiency.

We conclude that mutations in the HSD3B7 gene underlie C27 3ß-HSD deficiency, an autosomal recessive form of neonatal cholestasis. Preliminary data on long-term cholic acid therapy in children with this disorder show that it is an adequate and very effective treatment (11, 14, 15, 16). The identification of disease causing mutations will allow prenatal diagnosis within affected families and the initiation of cholic acid therapy in the immediate neonatal period.


   Acknowledgments
 
We thank the patients and their families for participation in this study, Daphne Davis and Nicole Raynaud for excellent technical assistance, Margrit Schwarz for initiating this collaboration, Jean D. Wilson and Jay D. Horton for critical reading of the manuscript, and the Falk Foundation for cholic acid.


   Footnotes
 
This work was supported by grants from the NIH (HL-20948, to D.W.R.), the Assistance Publique-Hospitaux de Paris (PHRC AOB 96026, to E. J.), and the NIH National Center for Research Resources (M0108084, to the Cincinnati Children’s Hospital General Clinical Research Center).

Abbreviations: 25-Hydroxycholesterol, [3H]cholest-5-ene-3ß,25-diol; C27 3ß-HSD, 3ß-hydroxy-{Delta}5-C27-steroid oxidoreductase; HEK, human embryonic kidney; OMIM, Online Mendelian Inheritance of Man.

Received October 10, 2002.

Accepted January 6, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Setchell KDR, Schwarz M, O’Connell NC, Lund EG, Davis DL, Lathe R, Thompson HR, Tyson RW, Sokol RJ, Russell DW 1998 Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7{alpha}-hydroxylase gene causes severe neonatal liver disease. J Clin Invest 102:1690–1703[Medline]
  2. Setchell KD, Suchy FJ, Welsh MB, Zimmer-Nechemias L, Heubi J, Balistreri WF 1988 D4-3-oxosteroid 5ß-reductase deficiency described in identical twins with neonatal hepatitis. A new inborn error in bile acid synthesis. J Clin Invest 82:2148–2157
  3. Clayton PT, Leonard JV, Lawson AM, Setchell KD, Andersson S, Egestad B, Sjovall J 1987 Familial giant cell hepatitis associated with synthesis of 3{alpha}, 7{alpha}-dihydroxy-and 3ß, 7{alpha}, 12{alpha}-trihydroxy-5-cholenoic acids. J Clin Invest 79:1031–1038
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