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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 7 2337-2342
Copyright © 1997 by The Endocrine Society

Clinical Studies

A Novel Splicing Junction Mutation in the Gene for the Steroidogenic Acute Regulatory Protein Causes Congenital Lipoid Adrenal Hyperplasia1

Eisaku Okuyama, Nozomu Nishi, Shoju Onishi, Susumu Itoh, Yoshiro Ishii, Hiroshi Miyanaka, Keinosuke Fujita and Yoshiyuki Ichikawa

Departments of Biochemistry (E.O., Y.I.), Endocrinology (N.N.), and Pediatrics (S.O., S.I., Y.I.), and Research Equipment Center (H.M.), Kagawa Medical University, Kita-gun, Kagawa 761–07; and the Department of Pediatrics, Osaka City General Hospital (K.F.), Toshima-ku, Osaka 534, Japan

Address all correspondence and requests for reprints to: Dr. Y. Ichikawa, Department of Biochemistry, Kagawa Medical University, Kita-gun, Kagawa 761–07, Japan. E-mail: yichikaw{at}kms.ac.jp


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Congenital lipoid adrenal hyperplasia (lipoid CAH) is a relatively common genetic disorder of adrenal and gonadal steroidogenesis and is the most severe form of CAH. As typical affected individuals cannot produce any steroid hormones or can only produce low levels of steroid hormones in the adrenals and gonads, including glucocorticoids, mineralcorticoids, and sex steroids, a genetic defect in the cholesterol side-chain cleavage enzyme, cytochrome P450scc (CYPXIA1), has been postulated to be the cause of their insufficient production to date. Recently, Lin and co-workers proved a link between mutations of the steroidogenic acute regulatory protein (StAR) gene and the lipoid CAH phenotype. Therefore, we investigated both the cytochrome P450scc and StAR genes in a Korean family with a fairly mild form of lipoid CAH to identify the mutation(s) causing this disease. The result was that no mutations could be found in the two genes, except for a thymine (T) insertion into intron 2 of the StAR gene, 3 bp from the splice donor site of exon 2. PCR-amplified StAR genes from a normal subject and the patient were cloned into an expression vector and then introduced into COS-7 cells. Northern blot and reverse transcriptase-PCR analyses indicated that the StAR messenger ribonucleic acid derived from the vector with the normal StAR gene spliced exons 2 and 3 correctly, whereas most, but not all, StAR messenger ribonucleic acid derived from the vector with the T-inserted StAR gene could not remove intron 2. We concluded from these results that the T insertion into the StAR gene accounts for the lipoid CAH phenotype in this patient.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CONGENITAL LIPOID adrenal hyperplasia (lipoid CAH), originally described by Prader and Gurtner (1), is the most severe form of CAH, involving impaired production of all steroid hormones, including glucocorticoids, mineralcorticoids, and sex steroids. Lipoid CAH results from deficient activity of the cholesterol side-chain cleavage enzyme (cytochrome P450scc), and the subsequent inability to produce pregnenolone results in the accumulation of cholesterol in the adrenal cortex (1, 2). The decreased concentration of one of above steroid hormones, cortisol, stimulates the secretion of ACTH through negative feedback. This increased ACTH secretion is a characteristic of this disease and causes adrenal hyperplasia (2). Clinically, the symptoms are adrenal insufficiency, poor feeding, lethargy, vomiting, diarrhea, hyponatremia, and hyperkalemia, and affected individuals are all phenotypically female regardless of karyotype. Furthermore, there may be increased pigmentation of skin creases, nipples, and genitalia. As a result, the patients die of salt loss, acidosis, and dehydration unless steroid hormone replacement therapy is begun immediately after birth.

The cholesterol side-chain cleavage enzyme, namely cytochrome P450scc (CYPXIA1), is the terminal enzyme of the cytochrome P-450-linked monooxygenase system (side-chain cleavage enzyme of cholesterol; EC 1.14.15.6), which catalyzes the conversion of cholesterol to pregnenolone on the matrix side of the mitochondrial inner membranes of the adrenal cortexes, ovaries, testes, placenta in a long pregnancy, and brain (3, 4, 5, 6, 7, 8), and is the initial rate-limiting reaction in the biosynthesis of steroid hormones other than renal ones (9). As the activity of the cytochrome P450scc linked monooxygenase system in adrenocortical mitochondria from patients with lipoid CAH was extremely low compared with that in normal subjects, it has been postulated that a deficiency of cytochrome P450scc causes this disease (10, 11, 12, 13). Therefore, many workers have focused on the detection of a mutation(s) in the cytochrome P450scc gene from affected individuals. However, no mutation has been found in this gene to date (14, 15, 16, 17).

LH and ACTH stimulate steroid biosynthesis (18, 19, 20) by increasing the transport of cholesterol from the outer mitochondrial membranes to the inner membranes. This process depends on a mechanism involving a cycloheximide-sensitive agent (21, 22, 23, 24, 25) and is the true regulated, rate-limiting step in the acute response of steroidogenic cells to ACTH stimulation (24).

Recently, a novel LH-induced mitochondrial protein, termed the steroidogenic acute regulatory protein (StAR), was purified from MA-10 mouse Leydig tumor cells (26). Extensive work has been carried out to characterize this protein (27, 28, 29, 30, 31). The details of this work can be summarized as follows. The StAR protein is expressed in steroidogenic organs, including the adrenals and gonads, but not in the placenta or brain. In the acute response of steroidogenesis to tropic hormone stimulation, the StAR protein is synthesized as a 37-kDa preprotein, which stimulates cytochrome P450scc activity, probably by acting on the outer mitochondrial membrane. Then, the StAR preprotein may be imported into the mitochondrial matrix and processed to the 30-kDa mature form, presumably to terminate its steroidogenic action. Recently, a number of mutations were detected in the StAR gene from patients with lipoid CAH (32, 33, 34). In other words, these reports proved an essential role for the StAR protein in steroidogenesis. In this report, we discuss the biological significance of a novel intronic mutation in the StAR gene.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patient

A patient from a Korean family diagnosed as having lipoid CAH according to the clinical and laboratory data was studied. She was the 3630-g, 52.5-cm product of a 40-week, 2-day gestation and thrived normally for 1 yr after birth without hormonal replacement therapy. It should be noted that as she was born without any abnormality, except for slight swarthiness, and seemed to thrive normally thereafter, a medical practitioner decided that her swarthiness was not severe enough for a close medical examination. However, about 1 yr of age her pigmentation became conspicuous, so she was hospitalized for inspection. The laboratory data are summarized in Table 1Go. Unlike typical laboratory data for lipoid CAH, the steroid hormone and electrolyte values in this patient were normal (see Discussion). However, ACTH and PRA were considerably high, with the result that she had hyperpigmentation of the skin. Furthermore, no response of the cortisol level to the administration of ACTH was observed (see Fig. 1Go). Taking into account these results, she was diagnosed as having lipoid CAH. At 3.5 yr of age, she was 15.6 kg and 97.6 cm, with 138 mEq/L Na, 4.0 mEq/L K, and 108 mEq/L Cl. Her parents are not consanguineous. Informed consent was obtained from the subject. This study was approved by the Department of Pediatrics, Kagawa Medical University.


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  		Table 1. Laboratory data of the patient

 


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  		Figure 1. Steroidal responses to ACTH (a), hCG and human menopausal gonadotropin (hMG) stimulations (b). Arrows in a, filled arrowheads, and open arrowheads in b indicate ACTH, hCG, and hMG stimulations, respectively. Stimulation tests were performed by administrating 0.25 mg synthetic ACTH (Cortrosyn Z), 2000 U hCG, and 30 U hMG, respectively.

 
Extraction of genomic DNA

Genomic DNAs were extracted from leukocytes of this patient, her mother, and healthy controls using a commercially available genomic DNA extraction kit, Sepa Gene (Sanko Junyaku Co., Tokyo, Japan), according to the manufacturer’s recommendations.

PCR amplification

We amplified the 5'-flanking regions, and all exons of the cytochrome P450scc and StAR genes by means of PCR amplification (35) to identify the mutation(s) causing lipoid CAH. For this amplification, oligonucleotides were originally synthesized based on the nucleotide sequence of the human cytochrome P450scc gene (36) and that of the StAR gene (28) (see Table 2Go).


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  		Table 2. Sequences and locations of oligonucleotides in the StAR gene that were used for amplification of exons, allele-specific PCR, and RT-PCR

 
The PCR protocol for this amplification comprised 1-min melting of the strands at 94 C, and then 1 min of denaturation at 94 C, 1 min of annealing at 55 C, and 2 min of extension at 72 C, using Taq DNA polymerase (Promega Co., Madison, WI), for 30 cycles. Final extension was then performed for 5 min at 72 C.

All of these reactions were performed with a Program Temp Control System PC-700 (Astec Co., Fukuoka, Japan).

Molecular cloning and sequencing

The PCR products described above were then cloned into the pT7Blue T-Vector (Novagen, Madison, WI). The sequencing reactions were performed by cycle sequencing, using a commercially available cycle sequencing kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS, Perkin-Elmer/Cetus, Norwalk, CT). The sequence analyses were performed using the ABI Prism 377 DNA Sequencer. We cloned and sequenced 10 clones/target to determine the genotype and to eliminate the PCR error(s). The nucleotide sequences were determined for both strands of the subcloned DNA for the sake of preciseness.

Analyses of DNA sequences

Analyses of the nucleotide sequences were carried out using the package, GENETYX. The nucleotide sequence of each clone described above was compared with those of the corresponding regions of the cytochrome P450scc and StAR genes previously reported (28, 36).

Construction of expression vectors

To construct expression vectors pcDNA-StAR and pcDNA-StAR(T), harboring the whole StAR genes from the normal subject and the patient with lipoid CAH, respectively, the PCR-amplified StAR genes were each digested with HindIII and then inserted into a pcDNA3 plasmid (Invitrogen Corp., San Diego, CA). The cloning junctions were verified by DNA sequencing analyses, and the plasmids were purified using commercially available DNA purification systems (Wizard Minipreps DNA Purification Systems, Promega Co.) before transfection. The oligonucleotides used for amplification of the whole StAR genes are listed in Table 2Go. The PCR protocol for this amplification comprised 1-min melting of the strands at 94 C, then 20 min of denaturation at 98 C, and 15 min of annealing and extension at 68 C, using Ex Taq (Takara Shuzo Co., Shiga, Japan), for 30 cycles. Final extension was then performed for 10 min at 72 C.

Transient expression of the StAR gene in COS-7 cells

COS-7 cells (simian virus 40-transformed monkey kidney) (37) were obtained from the Riken Gene Bank (Tsukuba Science City, Ibaragi, Japan). FBS was obtained from ICN Biochemicals Japan Co. (Osaka, Japan). Other reagents were obtained from Life Technologies (Gaithersburg, MD). COS-7 cells were cultured in DMEM supplemented with 10% (vol/vol) FBS and antibiotics (penicillin, 100 IU/mL; streptomycin, 100 mg/mL) at 37 C under a 5% (vol/vol) CO2 atmosphere. The cells were subcultured before transfection and then seeded into 35-mm plates at a density of 2 x 105 cells/plate. Transfection was carried out using a commercially available transfection reagent, TransIT (Takara Shuzo Co.), according to the manufacturer’s recommendations. The cells were transfected with either the pcDNA-STAR or the pcDNA-StAR(T) plasmid.

Ribonucleic acid (RNA) isolation

Total RNA was isolated from the transfected COS-7 cells cultured in 100-mm plates for 3 days using a commercially available nucleic acid and protein purification system, Isogen (Nippongene, Tokyo, Japan), according to the manufacturer’s recommendations. Briefly, the cells were lysed with Isogen and then scraped from the plates. RNA was extracted into the aqueous phase by treating the homogenate with chloroform and was precipitated with isopropanol. After quantification, the total RNA preparations were subjected to Northern blot analysis and reverse transcriptase-PCR (RT-PCR) analysis for StAR messenger RNA (mRNA; see Results).

Northern blotting

Northern blot analyses were carried out as described previously (38), using exon 2 of the human StAR gene as a probe.

Probe for Northern and Southern blotting

The probe, i.e. exon 2 of the human StAR gene, used for Northern and Southern blot analyses was labeled with digoxigenin-11-deoxy-UTP by means of PCR. The PCR protocol comprised 2-min melting of the strands at 94 C, then 1 min of denaturation at 94 C, 2 min of annealing at 60 C, and 2 min of extension at 72 C, using Taq DNA polymerase (Promega Co.) for 30 cycles. Final extension was performed for 8 min at 72 C. The amplified DNA fragment was purified by electrophoresis on 2.5% (wt/vol) agarose using a commercially available DNA retrieval system, Suprec-01 (Takara Shuzo Co.), according to the manufacturer’s recommendations.

RT-PCR

Total RNA isolated from transfected COS-7 cells was reverse transcribed using 20 mmol/L StAR-Rev primer by incubation for 40 min at 42 C with 20 U AMV RT XL (Life Science, Petersburg, FL), 25 U ribonuclease inhibitor (Toyobo Co., Tokyo, Japan), 1 mmol/L deoxy-NTPs, 25 mmol/L Tris-Cl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl2, and 2 mmol/L dithiothreitol in a final reaction volume of 30 mL. Then the complementary DNAs (cDNAs) were subjected to PCR amplification. The PCR protocol comprised 5-min melting of the strands at 95 C, then 1 min of denaturation at 95 C, sloping to 55 C over 2 min, 2 min of annealing at 55 C, and 2 min of extension at 72 C, using Taq DNA polymerase (Promega Co.), for 35 cycles. The final extension was performed for 5 min at 72 C. The oligonucleotides used for this analysis are listed in Table 2Go.

Southern blotting

Southern blot analysis was carried out as described previously (39) with the same probe as that used for Northern blot analysis.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Sequencing analyses

Although we investigated the cytochrome P450scc gene as well as the StAR gene of the patient, no mutation was found in either gene except for an insertion of T, 3 bp from the splice donor site of exon 2 of the StAR gene (Fig. 2Go). On account of this insertion, the preferred base changed from purine to pyrimidine (40). Furthermore, the patient and her mother were homozygous and heterozygous for this insertion, respectively, and none of the control subjects had this insertion. Taking into account these results, we hypothesized that the T insertion into intron 2 of the StAR gene disturbed the consensus splice donor sequence, 5'-GTA/G-3', with the result that intron 2 could not be removed from the mature StAR mRNA of the patient.



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  		Figure 2. Mutations in the StAR gene. The nucleotide sequence of the StAR gene from the patient is shown above the electropherogram. The insertion of T, 3 bp from the splice donor site of exon 2, is indicated in brackets. The exon is boxed, and the intron is dotted boxed.

 
Expression of the StAR gene in COS-7 cells

As adrenal or gonadal RNA of the patient was not available, we could not perform in vivo analysis on them. Therefore, to verify the above hypothesis, we first checked whether this T insertion influenced the processing of mRNA in COS-7 cells by Northern blot analysis. We constructed the pcDNA-StAR and pcDNA-StAR(T) plasmids, harboring the whole StAR genes from a normal subject and the patient, respectively, and transfected them into COS-7 cells. Exon 2 of the StAR gene was used as a probe in this analysis. If the StAR mRNA from the patient could not remove intron 2, the length of this mRNA must be about 300 bp longer than that from the normal subject. The result clearly indicated that the pcDNA-StAR(T) plasmid yielded a longer StAR mRNA than the StAR mRNA yielded by the pcDNA-StAR plasmid (see Fig. 3Go). It should be noted that the pcDNA-StAR(T) plasmid only yielded abnormally spliced StAR mRNA, as observed on Northern analysis.



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  		Figure 3. Northern blot analysis of the StAR mRNA. RNA samples (20 mg/lane) obtained from COS-7 cells were loaded as follows: lane 1, the normal subject; lane 2, the patient with lipoid CAH. The positions of 28S ribosomal RNA and 18S ribosomal RNA are indicated at the left of the blot.

 
RT-PCR

To determine the details of the abnormally spliced StAR mRNA, we performed RT-PCR analysis using the RNAs that were used for the Northern blot analysis as templates. For this analysis, we designed primers so that we might amplify the region around the junction of exons 2 and 3. The result was that the StAR mRNA from the patient was definitely 300 bp longer than that from the normal subject (see Fig. 4Go). For further confirmation, the 500- and 800-bp bands were each retrieved and subjected to sequencing analyses. The results indicated that the former exactly spliced exons 2 and 3, whereas the latter could not remove intron 2 (see Fig. 5Go). This unspliced intron 2 resulted in a premature termination codon. In this analysis, we noticed that not all of the StAR mRNA from the patient was abnormally spliced; some was spliced correctly. This was verified by Southern blot analysis (data not shown).



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  		Figure 4. RT-PCR analysis of the StAR mRNA. The RNAs that were used for Northern blot analysis were employed as templates. The PCR products were loaded in the same order as in Fig. 3Go. The standard size markers are indicated at the left with their lengths.

 


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  		Figure 5. Nucleotide sequences of the StAR cDNA clones. The nucleotide sequences of the cDNA clones and the putative amino acid sequences around the 3' terminus of exon 2 from the normal subject (A) and the patient with lipoid CAH (B) are indicated above the electropherograms. The cDNA clones were obtained from RT-PCR analyses.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We reported mutations in the genes for cytochrome P450scc and StAR in a Korean family with lipoid CAH. No mutations could be found in the two genes, except for the T insertion into intron 2 of the StAR gene. In general, mutations arising in introns have less significance than those arising in exons. However, from the following results, it can be reasonably assumed that the T insertion into intron 2 of the StAR gene accounts for the lipoid CAH phenotype in this patient. First, this T insertion is located 3 bp downstream from the splice donor site of exon 2. Although priority is given to a purine base (A or G) at this position (40), there was a pyrimidine (T) in place of purine in this case, with the result that the consensus splice donor sequence, 5'-GTPu-3', was disturbed. To confirm the effect of the T insertion on the processing of mRNA, we expressed the StAR genes from the normal subject and the patient in COS-7 cells. Figure 5Go clearly indicates that the StAR mRNA from the patient could not remove intron 2. As a result, the abnormally spliced mRNA could only translate defective StAR protein because of the premature termination codon in intron 2. In 1995, Lin (32), Tee (33), and their co-workers provided genetic evidence that a defect in the StAR protein could account for lipoid CAH. Although the StAR protein is synthesized as a 285-amino acid protein (41), our defective one was only 60 amino acids in length. This is much shorter than the above two cases. Therefore, it is certain that our defective StAR protein is not functional. Second, there is a correlation between the clinical diagnosis and the results of expression analysis of the StAR gene. Unlike the typical features of lipoid CAH, this patient exhibited normal steroid hormone and electrolyte values, and thrived normally for 1 yr after birth without hormonal replacement therapy (see Subjects and Methods). The features indicating lipoid CAH in this patient were a considerably high concentration of ACTH and hyperpigmentation. On Northern blot analysis of the StAR mRNA, we could detect only abnormally spliced StAR mRNA in the lane of the patient; however, RT-PCR analyses revealed trace of normally spliced StAR mRNA in the lane of the patient. These results suggest that the amount of this normally spliced StAR mRNA is barely enough to keep the levels of steroid hormones normal, and that a considerably high concentration of ACTH is necessary to produce an indispensable amount of normally spliced StAR mRNA. It follows from this that the patient had hyperpigmentation. Third, lipoid CAH is usually inherited in an autosomal recessive manner, and this is the case for the family we investigated. Allele-specific PCR analyses indicated that the patient was homozygous, her mother (and probably her father) was heterozygous, and the 17 normal subjects were not carriers of the T insertion (data not shown).

Certain oxygenated derivatives of cholesterol are effective steroidogenic substrates, because the greater solubility of these substrates might permit their transport to the inside of mitochondria, bypassing the process of cholesterol movement to cytochrome P450scc (42, 43, 44, 45, 46). Previous reports indicated that this process, i.e. the traverse of cholesterol from the outer to the inner mitochondrial membranes in response to ACTH stimulation, is dependent on de novo protein synthesis (21, 23, 24, 26, 27, 47), and this protein was termed StAR (26). As the StAR protein has nothing to do with the transport of oxygenated derivatives of cholesterol (32, 33), even with a deficiency of the StAR protein, adrenal and gonadal steroidogeneses seem to occur. On the contrary, if cytochrome P450scc does not function, steroidogenesis in these organs will not be able to occur at all, because even oxygenated derivatives of cholesterol as well as cholesterol must pass through cytochrome P450scc for steroidogenesis. The symptoms of lipoid CAH are considerably diversified; namely, some patients die soon after birth unless steroid replacement therapy is begun immediately, and others, including our patient, thrive for several months or more after birth without this therapy (10, 34). Therefore, we cannot rule out the possibility that mutations in the StAR gene are not the sole cause of lipoid CAH; in other words, mutations in the cytochrome P450scc gene can also account for this disease, although several reports proposed that mutations in the cytochrome P450scc gene were not the cause of lipoid CAH (14, 15, 16, 33). A previous report that a deletion in the cytochrome P450scc gene causes congenital adrenal hyperplasia in the rabbit supports this possibility (48). Previously, we examined patients with the serious lipoid CAH phenotype, who died within a few months of birth. This may be the case for a mutation(s) in the cytochrome P450scc gene. Therefore, we are now investigating cytochrome P450scc genes as well as StAR genes from the parents of those patients. Such studies will facilitate understanding of the cause of lipoid CAH.


   Acknowledgments
 
We are grateful to Drs. T. Ohnishi and H. Yoneyama for their helpful advice. We also thank an anonymous reviewer for the comments on an earlier draft of the paper.


   Footnotes
 
1 This work was supported in part by research grants (to Y.I. and N.N.) from the Ministry of Education, Science, and Culture of Japan. Back

Received November 25, 1996.

Revised February 4, 1997.

Revised March 13, 1997.

Accepted March 17, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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