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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 11 3983-3987
Copyright © 1999 by The Endocrine Society

Original Studies

A Novel Compound Heterozygous Mutation in the Steroidogenic Acute Regulatory Protein Gene in a Patient with Congenital Lipoid Adrenal Hyperplasia1

Noriyuki Katsumata, Yasusada Kawada, Yukiyo Yamamoto, Masanori Noda, Atsuko Nimura, Reiko Horikawa and Toshiaki Tanaka

Department of Endocrinology and Metabolism, National Children’s Medical Research Center (N.K., A.N., R.H., T.T.), Tokyo 154-8509; and the Department of Pediatrics, University of Occupational and Environmental Health (Y.K., Y.Y., M.N.), Kitakyushu 807-8555, Japan

Address all correspondence and requests for reprints to: Noriyuki Katsumata, M.D., Department of Endocrinology and Metabolism, National Children’s Medical Research Center, 3–35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Congenital lipoid adrenal hyperplasia (CLAH) is an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. Recently, it was reported that mutations in the steroidogenic acute regulatory protein (StAR) gene cause CLAH. In the present study, we have analyzed the StAR gene of a Japanese patient with CLAH. PCR amplification and subsequent nucleotide sequencing of the StAR gene and those of her parents revealed that the patient has a compound heterozygous mutation of this gene. In one allele, an undescribed G to C transversion in codon 217, which occurred at the last base of exon 5 and thus altered the splice donor site sequence, apparently resulted in a substitution of Arg to Thr (AGG to ACG: R217T), and in the other allele, a C to T transition in codon 218 caused a substitution of Ala to Val (GCG to GTG: A218V), which has been previously shown to abolish StAR activity. In vitro expression analysis of an allelic minigene that consists of exons 4–6 of the R217T mutant StAR gene showed that the G to C transversion in the splice donor site of exon 5 caused by the R217T mutation disrupts normal splicing, resulting in the complete skipping of exon 5, which alters the translation reading frame of exon 6, introduces a stop codon at amino acid position 174, and thus impairs the activity. A functional expression study of the R217T replacement mutant revealed that the mutant has no steroidogenesis-enhancing activity if the transcript of the R217T mutant allele is ever spliced normally and translated into the protein. From the genetic analysis of 50 healthy subjects, the novel R217T mutation was unlikely to be due to polymorphism. Together, these results indicate that this patient is a compound heterozygote for the mutation in the StAR gene (T217R and A218V) and that these mutations inactivate the StAR function and give rise to clinically manifest CLAH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CONGENITAL lipoid adrenal hyperplasia (CLAH), originally described by Prader and Siebenmann (1), is the most severe form of congenital adrenal hyperplasia, leading to impaired production of all steroids, including glucocorticoids, mineralocorticoids, and sex steroids. This disorder is inherited as an autosomal recessive trait, and the affected individuals are all phenotypically female with a severe salt-losing syndrome that is fatal unless treated with steroid replacement therapy (2). Because mitochondria from affected adrenal glands and gonads fail to convert cholesterol to pregnenolone, it had been postulated to be caused by a defect in the cholesterol side-chain cleavage enzyme, cytochrome P450scc (3, 4). However, no mutations have been revealed in the P450scc gene in affected individuals, suggesting a defect in another undefined factor involving conversion of cholesterol to pregnenolone (5, 6, 7).

Steroidogenic acute regulatory protein (StAR) is a 30-kDa phosphorylated protein that rapidly appears in the mitochondria of steroidogenic cells after tropic stimulation and is required in the acute regulation of steroidogenesis (8). It has recently been reported that mutations in the StAR gene cause CLAH (9). To date, 15 different mutations in the StAR gene have been found in patients with CLAH from various ethnic groups (9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

In the present study we analyzed the StAR gene in a Japanese patient with CLAH and found that the patient has a novel compound heterozygous mutation in the StAR gene that inactivates its function.


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

The patient is the first child of unrelated healthy Japanese parents. She was delivered at term after an unremarkable gestation through cesarean section because of breech presentation. Her birth weight was 2965 g. She was noticed to have hyperpigmentation, developed cyanosis and tachypnea soon after birth, and was referred to the neonatal intensive care unit of our university hospital. On physical examination she had remarkable pigmentation and normal female external genitalia with no ambiguity. The laboratory data are summarized in Table 1Go. Blood sugar was low, and plasma cortisol remained low despite markedly elevated ACTH. The electrolyte values were initially normal, but the infant developed vomiting and mild salt loss at 2 days of age (131 mEq/L sodium and 5.0 mEq/L potassium). She was diagnosed as having adrenal insufficiency. She was treated with hydrocortisone and fludrocortisone and did well. During her infancy testis-like structures became palpable in the lower inguinal region bilaterally, and the karyotype obtained at 7 months of age was 46,XY. At 3 yr of age the bilateral inguinal gonads were removed, and histological examination of the gonads confirmed the presence of testicular tissue. From these results, she was diagnosed as having CLAH.


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  		Table 1. Laboratory data of the patient

 
DNA amplification and sequence analysis of the StAR gene

The genomic DNAs of the patient and her parents were isolated from whole blood by proteinase K digestion and phenol/chloroform extraction after informed consent was obtained from each subject. DNA fragments that span each of seven exons of the human StAR gene were selectively amplified using PCR (19). The oligonucleotide primers and PCR conditions were the same as described previously (16). The amplified DNA fragments were purified by agarose gel electrophoresis and directly sequenced in both orientations by the dideoxynucleotide chain termination method (20) using a Thermo Sequenase kit (Amersham Pharmacia Biotech Japan Ltd., Tokyo, Japan).

Restriction analysis

The R217T mutation found in the patient and her mother, which creates a new restriction site for MaeII (Roche Molecular Biochemicals Gmbh, Mannheim, Germany), was confirmed by digestion PCR products with the restriction enzyme followed by electrophoresis on a 2% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME).

In vitro expression of the StAR minigene

Exons 4–6 of the mutant StAR gene carrying the R217T mutation and those of the normal StAR gene were amplified using the primer pair, S4-AS6, whose sequences were described previously (16). The respective PCR products were subjected to cloning into a pCRII plasmid using a TA cloning kit (Invitrogen, San Diego, CA), and the nucleotide sequences were confirmed by the dideoxynucleotide chain termination method. The inserts were cleaved from the plasmid DNA with a restriction enzyme EcoRI (Nippon Gene Co. Ltd., Tokyo, Japan) and then ligated to an expression vector pRK 5 (Fig. 3AGo). Four micrograms of the mutant or normal chimeric plasmid were transfected into COS-1 cells (RIKEN Cell Bank, Tsukuba, Japan) by the diethylaminoethyl-dextran method (21). After 48 h, total ribonucleic acid (RNA) was extracted from the cells with an ISOGEN kit (Nippon Gene Co. Ltd., Tokyo, Japan). The first strand complementary DNA (cDNA) was synthesized in a 12.5-µL reaction mixture containing 1 µg total RNA, 0.5 µg oligo(deoxythymidine)12–18, 0.8 mmol/L of each deoxy-NTP, 20 U ribonuclease inhibitor, 50 U TrueScript Reverse Transcriptase, and 1 x reaction buffer, which was supplied by the manufacturer (Sawady Technology Co. Ltd., Tokyo, Japan). The resulting cDNA was subjected to PCR amplification with a sense primer in exon 4 corresponding to nucleotides 442–462 (RTS4, 5'-GACAAAGTGATGAGTAAAGTG-3') and an antisense primer in exon 6 corresponding to the complement of 863–844 (RTAS6, 5'-TCGATGCTGAGTAGCCACTG-3'; Fig. 3AGo).



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  		Figure 3. Schematic presentation of the StAR minigene expression study. A, The genomic fragments from exons 4–6 of the R217T mutant and wild-type StAR gene are ligated to an expression vector pRK 5. The arrows below exons 4 and 6 denote the positions of the primers used in RT-PCR. B, Scheme of mRNA splicing of the StAR minigenes. The wild-type minigene is expected to yield an RT-PCR product of 422 bp, as shown in the left panel. Should exon 5 be skipped, a 237-bp product would be amplified by RT-PCR as shown in the right panel.

 
Functional expression of the R217T replacement mutant StAR

The wild-type StAR cDNA was amplified from a QUICK-Clone human adrenal gland cDNA (CLONTECH Laboratories, Inc., Palo Alto, CA) by PCR as reported previously (9), then ligated into the pCRII plasmid and sequenced. The R217T replacement was introduced in the wild-type StAR cDNA by the recombinant PCR method (22). The wild-type and R217T replacement mutant StAR cDNAs were cleaved from the plasmid DNA with EcoRI, then ligated to the expression vector pRK 5, and designated pRK-StAR and pRK-R217T, respectively. One microgram of the pRK-StAR, the pRK-R217T, or the empty pRK 5 plasmid was cotransfected into COS-1 cells with 1 µg F2 plasmid, which was constructed to express an NH2-P450scc-adrenodoxin reductase-adrenodoxin-COOH fusion protein (23) and was provided by Dr. Walter L. Miller (University of California, San Francisco, CA), as described above (21). In addition, 1 µg GH1-pRK, a human GH expression plasmid, was included in the transfection mixture and was used as an internal control for transfection efficiency. Forty-eight hours after transfection, the medium was collected for RIA of pregnenolone as described previously (24).

Western blot analyses of mitochondrial fractions isolated from transfected COS-1 cells were carried out with a polyclonal antibody raised against recombinant human StAR, which was provided by Dr. Jerome F. Strauss III (University of Pennsylvania, Philadelphia, PA) (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequencing analysis of the StAR gene

The entire coding region of the StAR gene from the patient and her parents, including the exon-intron boundaries, was directly sequenced. The sequencing analysis of the patient revealed two heterozygous mutations (Fig. 1Go). One mutation, designated R217T, was caused by a G to C transversion at the second nucleotide of codon 217 in exon 5, which apparently converted codon 217 (AGG) encoding Arg into ACG encoding Thr. The other, designated A218V, was caused by a C to T transition at the second nucleotide of codon 218 in exon 6, which changed codon 218 (GCG) encoding Ala to GTG encoding Val. The patient was also revealed to be homozygous for the D203A polymorphism (18). No other mutations were found in any of the seven exons or the exon-intron boundaries of the StAR gene from the patient.



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  		Figure 1. Partial nucleotide sequences of exon 5 (A) and exon 6 (B) of the StAR gene from the patient. The patient had an apparent heterozygous missense mutation at codon 217 and a heterozygous missense mutation at codon 218.

 
The sequencing analysis of her parents demonstrated that the father was heterozygous for the A218V mutation in the StAR gene, whereas the mother was heterozygous for the R217T mutation (data not shown).

Restriction analysis

As shown in Fig. 2AGo, the R217T mutation creates a new recognition site for the restriction enzyme MaeII. Thus, amplification of a 405-bp fragment from exon 5 of the StAR gene containing this mutation followed by digestion with MaeII should yield 325- and 80-bp fragments, whereas the normal fragment remains uncut. As shown in Fig. 2BGo, restriction analysis of the patient and the mother gave a mixed digestion pattern, confirming their heterozygosity, whereas for the father only the undigested 405-bp fragment was seen, demonstrating that he does not have the R217T mutation. Fifty normal subjects had only the uncut 405-bp fragment.



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  		Figure 2. Detection of G to C substitution in codon 237 by MaeII digestion. A, A 405-bp fragment containing exon 5 was amplified using the primer pair, S5-AS5, and subjected to MaeII digestion. The R217T mutation generates a MaeII site producing two fragments of 325 and 80 bp. B, Electrophoretic patterns of the digested PCR products from the patient, her parents, and normal controls. The patient and the mother had 325- and 80-bp fragments as well as a undigested 405-bp product. The father and normal controls had only the undigested fragment. {square} and {circ}, Normal controls; , the father bearing a heterozygous A218V mutation; , the mother bearing a heterozygous R217T mutation; , the patient bearing the compound heterozygous mutation.

 
Expression study of the StAR minigene

As shown in Fig. 3AGo, the R217T mutation destroys a consensus splice donor site sequence that exists at the exon 5-intron 5 boundary of the StAR gene. Therefore, we hypothesized that this mutation would cause abnormal splicing of the StAR gene. As adrenal or gonadal RNA of the patient was not available, we could not perform in vivo analysis of them to verify the hypothesis. Therefore, we made an in vitro expression study using the chimeric minigene system to analyze the transcription of the patient’s R217T mutant StAR gene. We first constructed two chimeric minigenes that consisted of an expression vector pRK 5 vector and the R217T mutant or wild-type StAR gene, which spans exons 4–6 (Fig. 3AGo). After the respective minigenes were expressed in COS-1 cells, the splice site selection patterns for exons 4–6 were assessed from the cDNA sizes that corresponded to the expressed messenger RNAs (mRNAs) by means of RT-PCR. If the mRNA splicing of the minigene proceeds normally, the RT-PCR amplification should yield a 422-bp product, whereas a 237-bp product would be amplified should the exon 5 be skipped (Fig. 3BGo). As shown in Fig. 4AGo, the RT-PCR product of the chimeric minigene that included the wild-type StAR gene was 422 bp as expected, whereas the transcript of the R217T mutant minigene was 237 bp, 185 bp shorter than that of the wild-type product. We confirmed nucleotide sequences of these two 422- and 237-bp products by direct DNA sequencing analysis. The former consisted of exons 4, 5, and 6, whereas the latter consisted only of exons 4 and 6 (Fig. 4BGo). This exon skipping altered the reading frame of exon 6, resulting in a premature stop codon (TAA) at codon 174, 57 bp downstream from the exon 4-exon 6 boundary.



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  		Figure 4. Demonstration of the effect of the R217T mutation on splicing of the StAR gene. A, Electrophoretic patterns of the RT-PCR products of the wild-type and mutant mRNAs. B, Partial nucleotide sequencing of the RT-PCR products. Note that exon 4 is followed by exon 6 because of the skipping of exon 5 in the mutant transcript.

 
Functional expression study of the R217T replacement mutant StAR

As the possibility that some mRNA was spliced normally in vivo could not be totally excluded because of unavailability of the patient’s tissues, we built the construct expressing the R217T replacement mutant StAR and assessed its activity in transfected cells. The wild-type StAR efficiently facilitated the conversion of cholesterol to pregnenolone, but the R217T replacement mutant failed to enhance the conversion (Table 2Go). Western blot analyses detected both the 37-kDa precursor and the 30-kDa mature form of the wild-type StAR and the R217T replacement mutant (data not shown).


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  		Table 2. Activity of wild-type and R217T replacement mutant StAR

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We identified a novel heterozygous mutation of the StAR gene in a Japanese patient affected with CLAH. Our patient had an undescribed R217T mutation in one allele and a previously reported A218V mutation in the other. The Q258X mutation of the StAR gene originally described by Lin et al. (9) is reported to account for about 70% of affected alleles in Japanese CLAH patients (11, 13). Nakae et al. reported one Japanese CLAH patient who has a homozygous frameshift mutation, 246insG, but does not have the Q258X mutation (13). Our patient seems to be the second Japanese CLAH patient who does not carry the common Q258X mutation.

The G to C transversion at the last base of exon 5 could result in a splicing error and/or a substitution of Arg to Thr at codon 217. Therefore, we constructed not only the chimeric StAR minigene originating from the R217T mutant allele to assess the splice site selection patterns, but also the R217T replacement mutant StAR cDNA to estimate the functional consequences of the replacement. The expression study of the chimeric StAR minigene demonstrated that the transcript of the R217T mutant minigene lacked exon 5. The skipping of exon 5 in the StAR mRNA also results in a frame shift in exon 6 that would, if translated, introduce 18 novel amino acids from codon 156 to 173 and a premature stop codon (TAA) at codon 174. It was demonstrated that the carboxyl-terminal 28 amino acids of the 285-amino acid StAR molecule is crucial for StAR activity by the functional expression study of the carboxyl-terminally truncated Q258X StAR mutant found in the CLAH patients. Thus, it is likely that the StAR mutant harboring exon 5 skipping caused by the R217T mutation would be functionally inactive even if translated. As expected, Tee et al. demonstrated that the mutant StAR mRNA lacking exon 5, which was caused by a T->A transversion in intron 4, does not encode a stable, functionally active protein by the functional expression study (10). The functional expression study of the R217T replacement mutant StAR demonstrated that the replacement of Arg to Thr at codon 217 resulted in total loss of the steroidogenesis-enhancing activity. Thus, we conclude that the R217T mutation completely abolishes StAR activity as the result of either the aberrant splicing or the single amino acid substitution.

The other mutation detected in our patient was the missense mutation A218V. This mutation has been found in Caucasian and Japanese patients with CLAH (11, 13, 16). The results of the expression study indicated that the A218V mutant shows minimal to null enhancement of the production of pregnenolone, although it can enter the mitochondria and be processed (11, 13). Thus, it is highly likely that this mutation as well as the R217T mutation is the cause of CLAH in this patient.

Our patient showed no virilization of the external genitalia and developed adrenal insufficiency soon after birth, indicating that both testicular and adrenal steroidogenesis had been severely impaired when she was born. These findings are in good agreement with the results of the in vitro expression study demonstrating that the R217T and the A218V mutations cause total loss of StAR activity. It is noteworthy that the CLAH patient reported by Tee et al. (10) had a relatively late onset of symptoms (8 weeks of age). In this patient the T->A transversion in intron 4 also caused the splicing error eliminating exon 5 in most of StAR mRNA, but some StAR mRNA was spliced normally; thus, StAR activity was presumably retained in part.

In conclusion, we have identified a novel genetic mutation in the StAR gene that results in CLAH and demonstrated that there exists some relationship between symptoms and StAR activity in CLAH patients.


   Acknowledgments
 
We thank Dr. Walter L. Miller (University of California, San Francisco, CA) for providing the F2 plasmid, and Dr. Jerome F. Strauss III (University of Pennsylvania, Philadelphia, PA) for providing the antihuman StAR serum. We are also grateful to Ms. Shoko Mikami, Ms. Atsuko Nagashima-Miyokawa, and Ms. Mitsuko Sakai for technical assistance.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, and Culture, Japan; a Grant for Pediatric Research (10C-3) from the Ministry of Health and Welfare, Japan; and a Grant for Liberal Harmonious Research Promotion System from the Science and Technology Agency, Japan. Back

Received March 2, 1999.

Revised July 13, 1999.

Accepted July 26, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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