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Congenital Lipoid Adrenal Hyperplasia Caused by a Novel Splicing Mutation in the Gene for the Steroidogenic Acute Regulatory Protein

Department of Endocrinology (A.A.G., C.A.C., L.M.M., C.E.F.), Pediatrics (M.L.R., P.B.), and Pathological Anatomy (A.S.), Faculty of Medicine; and Department of Molecular Genetics and Microbiology, Faculty of Biological Sciences (J.A.T., A.V.), Pontificia Universidad Católica de Chile, 114-D Santiago, Chile

Address all correspondence and requests for reprints to: Carlos E. Fardella, Department of Endocrinology, Faculty of Medicine, Pontifica Universidad Católica de Chile, Lira 85, piso 5 Santiago, Chile. E-mail: cfardella{at}med.puc.cl.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Steroidogenic acute regulatory protein (StAR) plays a crucial role in the transport of cholesterol from the cytoplasm to the inner mitochondrial membrane, facilitating its conversion to pregnenolone by cytochrome P450scc. Its essential role in steroidogenesis was demonstrated after observing that StAR gene mutations gave rise to a potentially lethal disease named congenital lipoid adrenal hyperplasia, in which virtually no steroids are produced. We report here a 2-month-old female patient, karyotype 46XY, who presented with growth failure, convulsions, dehydration, hypoglycemia, hyponatremia, hypotension, and severe hyperpigmentation suggestive of adrenal insufficiency. Serum cortisol, 17OH-progesterone, dehydroepiandrosterone sulfate, testosterone, 17OH-pregnenolone, and aldosterone levels were undetectable in the presence of high ACTH and plasma renin activity levels. Immunohistochemical analysis of testis tissues revealed the absence of StAR protein. Molecular analysis of StAR gene demonstrated a homozygous G to T mutation within the splice donor site of exon 1 (IVS1 + 1G>T). Her parents and one brother were heterozygous for this mutation. In vitro analysis of the mutation was performed in COS cells transfected with minigenes coding regions spanning exon-intron 1 to 3 carrying the mutant and the wild-type sequences. RT-PCR analyses of the mutant gene showed an abnormal mRNA transcript of 2430 bp (normal size 433 bp). Sequence analysis of the mutant mRNA demonstrated the retention of intron 1. Immunolocalization of the StAR minigene product detected the peptide in the mitochondria of COS cells transfected with the wild-type minigene but not in those transfected with the mutant minigene. We conclude that this mutation gives rise to a truncated StAR protein, which lacks an important N-terminal region and the entire lipid transfer domain.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
CONGENITAL LIPOID ADRENAL hyperplasia (CLAH) is a severe autosomal recessive form of congenital adrenal hyperplasia. It is characterized by the complete absence of steroid hormone biosynthesis, including glucocorticoids, mineralocorticoids, and sex steroids. As a result, 46XY males have female genitalia, and affected individuals die due to salt loss, hyperkalemic acidosis, and dehydration unless steroid hormone replacement therapy is begun immediately after birth (1, 2).

In CLAH, cholesterol cannot be metabolized to pregnenolone and cortisol synthesis is interrupted, causing an increase of ACTH release, which stimulates cholesterol recruitment and its accumulation in lipid droplets of steroidogenic cells. This lipid accumulation swells the cell and damages its cytoarchitecture and, consequently, its steroidogenic capacity (2).

Genetic analyses of CLAH patients have demonstrated that mutations in the gene for the steroidogenic acute regulatory protein (StAR) are responsible for this condition (3). This protein triggers the rapid delivery of substrate-cholesterol from the outer to the inner mitochondrial membrane of adrenal and gonadal steroidogenic cells (4), the rate-limiting step in the steroidogenic acute response to trophic hormone stimulation (5, 6). It is now clear that the preprotein of 37 kDa is the active form that determines the activity of the StAR protein, which is proportional to its residency time in the outer mitochondrial membrane (7).

Thirty mutations in the StAR gene have been described as causing CLAH, including missense, nonsense, and frame-shift mutations (2, 8). Most of these mutations are located between exons 5–7 and affect the critical StAR-related lipid transfer (START) domain (8). Mutations in the START domain drastically affect the activity of StAR; indeed, deletion of just 28 carboxyl-terminal residues suppresses all activity (3, 9). On the other hand, deletion of the 62 residues encoding the mitochondrial leader (N-terminal domain) of StAR protein precludes the mitochondrial entry, but it does not affect its activity (10), suggesting that StAR acts on the outer mitochondrial membrane.

To date, four splice-site mutations in the StAR gene have been reported in patients with CLAH (11, 12, 13, 14). In the present study, we analyzed the molecular consequences of a novel splice-junction mutation in the StAR gene, found in a Chilean patient with CLAH.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

A 2-month-old female patient was referred to our hospital for growth failure, convulsions, dehydration, hypoglycemia, hyponatremia, hypotension, and severe hyperpigmentation that suggested adrenal insufficiency. She had normal female external genitalia with no ambiguity. Adrenal steroids in serum were very low or undetectable: cortisol, 16.8 nmol/liter [normal value (NV), 140–690 nmol/liter)]; aldosterone, 55.4 pmol/liter (NV, 140–560 pmol/liter); dehydroepiandrosterone sulfate, 0.78 µmol/liter (NV, 1.3–6.8 µmol/liter); and testosterone, 0.35 nmol/liter (NV, 10–35 nmol/liter). 17-OH Progesterone and 17-OH pregnenolone were undetectable. Plasma renin activity and plasma ACTH levels were extremely high, 57.0 ng/ml·h (NV, 1–2.5 ng/ml·h) and 101.2 pmol/liter (NV, 2–11 pmol/liter), respectively. The karyotype was 46XY. Testis-like structures were palpable in the bilateral lower inguinal region, and the histological appearance showed testicular tissue. Thus, this patient was diagnosed as having CLAH. Her parents and one brother were healthy. The parents denied consanguinity or inbreeding.

DNA amplification and sequence analysis of the StAR gene

Genomic DNA was isolated from peripheral leukocytes of the index case, parents, brother, and 100 healthy controls using a commercially available DNAzol reagent (Invitrogen Corp., San Diego, CA). PCR amplification of StAR gene spanning seven exons and their respective introns was performed in five segments using the oligonucleotide primers shown in Table 1. PCR conditions included 5 min at 94 C (initial denaturation), followed by 30 cycles (1 min at 94 C, 1 min at 56 C, and 2 min at 72 C) and 10 min at 72 C (final extension) using 1.5 units Taq DNA polymerase (Invitrogen). Sequence analysis of StAR gene was performed in an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequences were matched with the StAR gene published in GenBank (U29099-U29105) (Ref.15) and the online program BLAST software (16).

A restriction analysis was performed to confirm the mutation found in the StAR gene and to determine the haplotype of the patient, her family, and 100 healthy controls. The restriction endonuclease HphI (New England Biolabs, Beverly, MA) recognizes the normal sequence GGTGA(N)₈ and cuts the normal 299-bp PCR product into two fragments of 198 and 101 bp. The G to T mutation in intron 1 (IVS1 + 1G>T) changes the recognition site for the restriction endonuclease HphI from G/GTGA to G/TTGA. Thus, the PCR product of 299 bp (exon1-intron1 boundary region) is protected from cleavage when the sequence is mutated. Analyses were performed using 0.5 U of HphI incubated for 16 h at 37 C. Restriction assay products were electrophoresed in 6% polyacrilamide gel and visualized with ethidium bromide.

In vitro expression of StAR minigene

We did not perform in vivo analysis of StAR mRNA because we were unsuccessful in obtaining fresh adrenal or gonadal tissue, but we performed an in vitro study using StAR minigenes. The minigenes included the region between exons 1–3, carrying the mutant and wild-type gene sequences. Minigenes were obtained from genomic DNA using the exon (EX) flanking primers EX 1 and EX 2–3R (Table 1). PCR amplification products were cloned into pGEM-T Easy vector (Promega Corp., Madison, WI), and positive clones were subcloned in pCR3 expression vector (Invitrogen). The pCR3 clones containing the mutated and normal minigene were confirmed by sequencing analysis and transfected into COS cells using LipofectAMINE 2000 reagent (Invitrogen). COS cells were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum and antibiotics (penicillin, 100 IU/ml; streptomycin, 100 mg/ml) at 37 C and 5% CO₂ atmosphere. After 48 h, total RNA was extracted with TRIZOL LS reagent (Invitrogen). Single-strand cDNA was synthesized from total RNA by RT-PCR using the SuperScript II Kit (Invitrogen). An aliquot of the reaction mixture was heated at 95 C for 5 min and then added to a PCR. The PCR conditions to amplify cDNA were similar to those previously described in the StAR gene amplification, and the primers used were EX1 F (Table 1) and EX 3 R (5'-AAGAAGGAGAGTCAGCAGG-3').

Immunohistochemical localization of StAR

Formalin-fixed paraffin-embedded testis sections (5 µm) of a previous biopsy were stained with hematoxylin-eosin and subjected to observation in a light microscope. StAR protein expression was visualized on tissue sections with peroxidase-conjugated antibodies (Automated Immunodetection System, BioGenex, San Ramón, CA). Briefly, sections were incubated in 3% H₂O₂ to block endogenous peroxidase activity. Nonspecific binding was avoided by incubating with goat serum diluted 1:10 in PBS 4% BSA. The sections were incubated with rabbit polyclonal anti-StAR antibody diluted 1:200. Tissue sections were then washed and incubated with peroxidase-conjugated goat antirabbit antibody (1:200). The slide was developed with 3,3'-diaminobenzidine tetrahydrochloride (BioGenex).

Immunocytochemistry of StAR

We performed an immunocytochemical study with StAR antiserum to evaluate the subcellular localization (i.e. mitochondria) of the resulting wild-type gene construct, StAR N-110 (110 residues in length), or the mutant construct (mutant peptide). COS cells transfected with the wild-type or mutant minigene were incubated with MitoTracker Red CMXRos reagent (Molecular Probes, Inc., Eugene, OR) to identify mitochondria (red emission) and fixed using a methanol-acetone (50:50) solution 3 min at 20 C. Cells were washed and incubated with blocking solution consisting of 0.2% porcine gelatin (Sigma, St. Louis, MO). Subsequently, they were incubated with StAR antiserum (1:200) 18 h at 4 C (available StAR antiserum recognizes from residue 62 to 285 of the wild-type StAR), washed, and incubated with fluoroscein isothiocyanate (FITC)-conjugated goat antirabbit serum (1:200) with green emission (Bio-Rad, Hercules, CA). Cover glasses containing cells were mounted on slides with Fluoromount G (Emsdiasum, Fort Washington, PA) and visualized in a Cannon fluorescence microscope and Zeiss Axio Vert 200M confocal microscope (Carl Zeiss, Inc., Thornwood, NY). MitoTracker Red (Molecular Probes, Inc.) and FITC fluorescence channel were selected to perform the visualization of mitochondria and StAR protein in COS cells.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Molecular analysis of the StAR gene

The entire coding regions of the StAR gene from the patient, including the exon-intron boundaries, were sequenced. The sequencing analysis of the patient revealed a homozygous G to T mutation at the first nucleotide of intron 1 (IVS1 + 1G>T) (Fig. 1A). No other mutations were found in any of the seven exons or the exon-intron boundaries of the StAR gene.

View larger version (56K): [in this window] [in a new window]   FIG. 1. A, Sequencing analysis of the StAR gene in the patient and a control. Underlined base shows the homozygous mutation (IVS1 + 1G>T) one nucleotide from splice donor site of exon 1 (top sequence) and the normal consensus sequence G/GT (bottom). B, Restriction analysis. A 6% polyacrilamide gel electrophoresis shows the PCR products digested with the restriction endonuclease HphI in the patient, mother, father, brother, and a healthy control. The G to T mutation avoids the cleavage of 299-bp PCR product from restriction (see Patients and Methods for details).

To confirm the IVS1 + 1G>T mutation, we performed a restriction analysis. As shown in Fig. 1B, the patient had only the noncleaved 299-bp fragment, indicating that the mutation was present in both alleles. Both parents and one brother showed the original 299-bp fragment and two other bands in 194 and 105 bp, confirming that they were heterozygous for the intronic mutation (see Patients and Methods for details). Enzymatic restriction in normal subjects showed only 194- and 105-bp fragments, indicating the presence of the normal sequence (only one representative subject is shown in Fig. 1B).

Expression of the StAR minigene in COS cells and RT-PCR

Functional consequences of the mutation were analyzed in transfected COS cells. Amplification of cDNA from mutant StAR minigene sized 2430 bp, whereas cDNA from normal StAR minigene sized 433 bp. The 433-bp fragment represents the normally spliced coding region (exons 1–2–3) (Fig. 2, A and B). The longer PCR product (2430 bp) includes the normally spliced coding regions and the unspliced intron 1. The retained intron 1 adds 1997 bp to the mutant PCR product, explaining its abnormal size. For further confirmation, the 2430-bp RT-PCR product was subjected to sequencing analysis (Fig. 2C) to demonstrate the presence of the aberrant splicing site in the 5'-end of intron 1 (G/TT) and the appearance of a stop codon (UGA) approximately 50 nucleotides downstream.

View larger version (59K): [in this window] [in a new window]   FIG. 2. In vitro expression of StAR minigene in COS cells. A, A 1% agarose gel showing the electrophoretical pattern of RT-PCR products amplified from RNA obtained from COS cells transfected with either the wild-type (433 bp) or the mutant minigene (2430 bp). Human testis was used as control to identify the normal RT-PCR product. B, Consensus splice sequence G/GT is disrupted by the G to T mutation (*) causing retention of intron 1. C, Electropherogram showing the nucleotide sequence of StAR cDNA from COS cells transfected with mutant minigene. Retention of intron 1 originates a new stop codon (UGA) approximately 50 bp downstream from the splicing site. WT, Wild-type; MUT, mutant; MW, molecular weight.

Light microscopy revealed that testis was composed of seminiferous tubules with spermatogonia and Sertoli cells (Fig. 3A). The interstitial space showed loss of connective tissue and abundant fibroblast-like cells (Fig. 3A) and Leydig cells (LC). The presence of LC was confirmed by electronic microscopy analysis (data not shown). In the control, testis-specific StAR immunoreactivity was observed in LC (Fig. 3B), but it was absent in the patient’s testis sample (Fig. 3C).

View larger version (88K): [in this window] [in a new window]   FIG. 3. Light microscopy appearance of the 2-month-old patient (C) and control (A and B) testis. The seminiferous tubules are composed by spermatogonia (S) and Sertoli cells (SC). A, The spermatogonias show a light nuclear texture, and immature SC show an oval and elongated nucleus. In the interstitium (I), there is loss of connective tissue and abundant fibroblast-like cells. B, Immunohistochemistry in the control testis tissue identified StAR protein in the cytoplasm of LC in a granular pattern (arrow). Unspecific immunoreactivity is observed in seminiferous tubules (*). C, Immunohistochemistry in the patient testis did not show StAR protein in LC. D–O, Immunocytochemistry localization of StAR protein in COS cells. Cells were grown and incubated with MitoTracker Red (Molecular Probes) CMXRos (E, I, M) and anti-StAR antiserum-FITC (F, J, N). In wild-type (WT) transfected COS cells, the FITC signal indicates the presence of StAR N-110 (F). Image overlay in WT shows colocalization of mitochondria and StAR N-110 peptide (G). Mutant (Mut) and untransfected cells (UC) do not have any FITC signal (J, N), showing that there is not StAR peptide. Scale bar, 10 µm.

The subcellular localization of the StAR mutant peptide was evaluated by immunocytochemical assays. We identified the presence of StAR N-110 peptide only in COS cells transfected with the normal minigene (FITC) signal; Fig. 3F). We did not observe any FITC signal in the mutant minigene-transfected cells (Fig. 3J). The StAR protein colocalized with mitochondria only in COS cells transfected with the wild-type minigene (yellow signal) (Fig. 3G).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In this study, we identified a novel intronic homozygous mutation (IVS1 + 1 G>T) of the StAR gene in a Chilean patient affected with CLAH. No consanguinity or inbreeding was determined after several clinical interviews with her parents, both Chilean natives with Spanish and Italian ancestry. CLAH is a rare disorder, but with a high prevalence (5–10%) in some races such as Japanese (2) and Palestinian (2, 17), in whom it is autosomal-recessive. Few molecular and clinical studies have been carried out in the Latin American population, except for two reported cases, a Mexican subject (2) and a Guatemalan subject (8).

In our patient, the G to T mutation at the first base of intron 1 disrupts the consensus sequences (18, 19, 20, 21), critical for splicing (original donor sequence is G/GT that changed to G/TT in mutant gene), thereby retaining intron 1 and producing a nonfunctional protein. RT-PCR from transfected COS cells demonstrated that StAR cDNA from the mutant gene had intron 1, which generated a mRNA larger than the normally spliced wild-type minigene (StAR N-110) or control (testis tissue) cDNA (Fig. 2A). Splicing site mutations in the StAR gene have been reported previously (12, 13, 14). Recently, Achermann et al. (14) described a homozygous mutation within the splice donor site of exon 1 (IVS1 + 2 T>G) that affects also the consensus sequence of the splice donor site.

Sequence analysis of the mutant cDNA suggests that mutant mRNA would be translated until approximately 50 bp from the beginning of the intron 1, because a stop codon (UGA) localized at this point interrupts the translation (Fig. 2C). However, we found three more alternative translation start sites (TSS) in the mutant mRNA; two are located in methionines 17 and 20 of wild-type StAR (22), and the third appears 24 bp downstream of the splice donor site of exon 1 (Fig. 2C). These alternative TSS keep the same reading frame of the original peptide, maintaining the stop codon within intron 1. In 1995, Gradi et al. (22) described two different StAR preproteins: the wild-type preprotein, and a shorter form produced from translation at methionine 20, which supports the theoretical appearance of a putative TSS in the mutant mRNA. Because intron 1 is too large, there could be other TSS downstream of intron 1, most likely giving rise to a nonfunctional protein with unspecific activity.

The StAR protein has 26 residues that form an amphipathic -helix in the N-terminal region and are characteristic of proteins imported into mitochondria (5, 22, 23). In the StAR protein, 19 of them would be necessary for its correct folding and activity (10). The mutant StAR minigene peptide, with the normal TSS at methionine 1 and the abnormal stop codon, contains 21 of 26 original N-terminal residues, which could promote the mitochondrial destination of this truncated peptide. However, preliminary structural modeling studies using the 3D-PSSM web server (24) revealed that the normal amphipathic -helix of the mitochondrial leader sequence is changed to a ß-sheet secondary structure, which is not present in proteins imported into mitochondria. Indeed, colocalization of the wild-type StAR N-110 with mitochondria supports a right splicing and translation of exons 1–3, with an adequate protein folding (Fig. 3F). On the other hand, the absence of FITC signal in mitochondria of COS cells transfected with the mutant construct could be explained by either the absence of the mutant peptide or the presence of an unrecognized peptide by the available antibody (shorter than N-terminal 62 residues). Immunohistochemical analysis in the patient’s gonadal tissue also revealed the absence of StAR protein in LC (Fig. 3C).

In summary, we report a patient with CLAH associated to a novel homozygous mutation localized in intron 1 (IVS1 + 1 G>T) of the StAR gene. This mutation produces an aberrant splicing of primary RNA, a nonfunctional mRNA, and, finally, the truncation of most of the StAR protein, including the N-terminal region and START domain, turning off the StAR activity and promoting the cholesterol accumulation.

	Acknowledgments

 
We thank Dr. J. F. Strauss III (University of Pennsylvania Medical Center) for the rabbit polyclonal antihuman StAR antiserum, and Drs. Manuel Santos and Andrés Toro (Department of Molecular Cell Biology, Faculty of Biological Science, Pontificia Universidad Católica de Chile) for the technical assistance.

	Footnotes

 
This work was supported by el Fondo Nacional de Investigacion Cientifica y Tecnologica de Chile Proyecto 1011035.

Abbreviations: CLAH, Congenital lipoid adrenal hyperplasia; FITC, fluoroscein isothiocyanate; LC, Leydig cell(s); NV, normal value; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; TSS, translation start site(s).

Received February 27, 2003.

Accepted October 13, 2003.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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