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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 43-48
Copyright © 2004 by The Endocrine Society

Special Feature

Two Intronic Mutations Cause 17-Hydroxylase Deficiency by Disrupting Splice Acceptor Sites: Direct Demonstration of Aberrant Splicing and Absent Enzyme Activity by Expression of the Entire CYP17 Gene in HEK-293 Cells

Marivania Costa-Santos, Claudio E. Kater, Eduardo P. Dias and Richard J. Auchus

Division of Endocrinology and Metabolism, Department of Medicine, Escola Paulista de Medicìna, Federal University of Sao Paulo (M.C.-S., C.E.K.), Sao Paulo, Brazil 04039-034; Felício Rocho Hospital (E.P.D.), Belo Horizonte, Brazil 30130-100; and Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center (R.J.A.), Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: Richard J. Auchus, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: richard.auchus{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
To date, only two among 46 mutations in the CYP17 gene cause 17-hydroxylase deficiency (17OHD) by disrupting mRNA splice donor sites. We studied two subjects with intronic CYP17 mutations: a compound heterozygote for Y329D plus an AG to CG substitution at the 3' end of intron 2, and a homozygote for a TTTT deletion near the 3' end of intron 3. We hypothesized that both mutations caused 17OHD by disrupting splice acceptor sites. To prove this mechanism, the entire CYP17 genes (wild type and both mutations) were amplified, subcloned into pcDNA3, and expressed in HEK-293 cells. The mRNA derived from the wild-type CYP17 gene was correctly spliced and translated into active enzyme, as shown by the correct sequence in the RT-PCR products and by the 17-hydroxylation of progesterone. In contrast, cells expressing the mutant genes had no 17-hydroxylase activity. The mRNA derived from the AG to CG mutation used the first AG in exon 3 as the splice acceptor site, shifting the reading frame and introducing a stop codon. RNA derived from the TTTT deletion skipped exon 4 entirely, deleting 29 amino acids in-frame. Our data show that these are the first two 17OHD cases resulting from mutations that alter splice acceptor sites. These studies also demonstrate the feasibility of expressing the entire CYP17 gene, with simultaneous protein and RNA analysis, as a general methodology for characterizing how intronic CYP17 mutations cause 17OHD.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
17-HYDROXYLASE DEFICIENCY (17OHD) is an uncommon disease, with an estimated incidence of 1:50,000 (1). Classical features include hypertension and hypokalemia with renal potassium wasting, low cortisol, and suppressed plasma renin activity. In addition, 17OHD subjects show a lack of pubertal development with low sex steroids, elevated gonadotropins, and high progesterone concentrations. Similar to other enzyme deficiencies, the diagnosis of 17OHD is confirmed by the consistent elevation of the immediate precursors to the enzymatic block, 11-deoxycorticosterone and corticosterone, as well as 18-hydroxydeoxycorticosterone and 18-hydroxycorticosterone (2).

The CYP17 gene, located on chromosome 10q24-q25, consists of 8 exons and 7 introns and spans a total of 6.4 kb (3, 4). The same 2.4-kb mRNA is expressed in both the adrenals and gonads (5). At this writing, 44 mutations affecting the coding regions have been described (6, 7, 8, 9, 10, 11). In addition, 2 mutations in splice donor sites have been reported in intron 2 (position +5 G->T) (12, 13) and in intron 7 (position +5 G->A) (14), which result in the skipping of exons 2 and 7, respectively. In the present study we describe the first 2 mutations affecting CYP17 splice acceptor sites. We also provide direct evidence that the mutations disrupt mRNA splicing and that negligible active enzyme is produced from the alleles with intronic mutations.


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

Subject 1 is a 27-yr-old, 46,XX Brazilian who experienced spontaneous sexual development (Tanner V) and irregular menses. At the age of 17 yr, she underwent bilateral oophorectomy due to large, persistent ovary cysts and pelvic pain. The presence of hypertension (170/100 mm Hg), hypokalemia (2.8 mEq/liter), and elevated serum progesterone concentrations after oophorectomy suggested the diagnosis of 17OHD, which was confirmed by finding elevated basal and ACTH-stimulated values of 17-deoxysteroids (Table 1Go). This subject gave written informed consent for hormone measurements (performed at Nichols Diagnostics, San Juan Capistrano, CA) and DNA studies.


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  		TABLE 1. Laboratory data for subjects

 
Subject 2 was a 46,XY American affected by severe hypertension and hypokalemia since her childhood in the late 1960s. The diagnosis of 17OHD was based on extremely elevated 11-deoxycorticosterone and corticosterone values (Table 1Go; courtesy Dr. Edward Biglieri, San Francisco General Hospital, San Francisco, CA) and failure of pubertal progression, and skin fibroblasts were obtained as part of her evaluation. Intraabdominal testes were removed by laparotomy, and her blood pressure remained elevated with erratic compliance to therapy. She died after complications of a stroke.

Identification of mutations

Genomic DNA (500 ng) extracted from leukocytes (subject 1) or skin fibroblasts (subject 2; Pure Gene DNA Isolation D5000, Gentra Systems, Inc., Minneapolis, MN) was amplified in a final volume of 50 µl with 3% dimethylsulfoxide, 0.2 mM deoxy-NTPs, and 0.4 µl TaKaRa ExTaq polymerase (Takara, Shuzo Co., Shiga, Japan) using the buffer from the manufacturer as previously described (11). Oligonucleotide primers c17geneS1 and c17geneAS (Table 2Go) were used for PCR amplification of DNA from subject 1 using 40 cycles of 1 min at 94 C for denaturation, 30 sec at 60 C for annealing, and 5 min at 70 C for extension. For subject 2, DNA was amplified in two PCR reactions using primer sets 1FEco + I4AS1 and 1RHind + I3S1 (Table 2Go); the conditions were 40 cycles of 30 sec at 95 C for denaturation, 1 min at 60 C for annealing, and 5 min at 68 C for extension. The amplicons were gel-purified and submitted for direct sequencing of the exons and flanking intronic DNA by the dye termination method on a PE Applied Biosystems instrument (McDermott Center Sequencing Facility at University of Texas Southwestern Medical Center, Dallas, TX). The sequence was and compared with GenBank sequence (access no. M19489) for CYP17 (3) using MacVector 6.5.3 (Accelrys Corp., San Diego, CA). Both mutations were confirmed with a second PCR reaction and sequencing in the opposite direction.


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  		TABLE 2. Oligonucleotide primers used for genomic DNA amplification and sequencing(a), construction of mutations in pcDNA3(b), and RT-PCR(c)

 
Construction of expression plasmids for CYP17 genes

The complete genes (wild-type and mutations) were constructed by amplification of genomic DNA in two pieces and cloning sequentially into the mammalian expression vector pcDNA3. Genomic DNA (1 µg) was amplified in 100-µl final volume with 3% dimethylsulfoxide, 0.2 mM deoxy-NTPs, and 0.4 µl ExTaq. The primers for the amplification were 1FHind and I4AS2 (PCR A), and 1REco and I3S3 (PCR B; Fig. 1Go and Table 2Go). PCR conditions were 40 cycles of 1 min at 94 C for denaturation, 1 min at 65 C for annealing, and 3 min at 70 C for extension. The product of PCR A, encompassing exons 1–3, introns 1–2, and part of intron 3, was digested with HindIII (5') and EcoRI (3'), gel-purified, and directionally ligated into vector pcDNA3 (Invitrogen, Carlsbad, CA). The PCR B product, containing exons 4–8, introns 4–7, and part of intron 3, was digested with EcoRI, gel-purified, and ligated into the EcoRI site of the intermediate plasmid containing only PCR A.



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  		FIG. 1. Construction and subcloning of CYP17 mutations AG to CG and TTTT deletion. The complete CYP17 genes (wild type, AG to CG, and TTTT deletion mutations), including all exons and introns, were constructed in two steps (PCR-A and PCR-B) and sequentially cloned into pcDNA3 using HindIII (5') and EcoRI (3') restriction enzymes. The DNA amplification was designed to overlap the internal EcoRI restriction site in intron 3. Because subject 1 was a compound heterozygote for the AG to CG mutation in intron 2 and Y329D in exon 6, the wild-type CYP17 gene was used as a template for PCR-B for that construct.

 
The orientation of the PCR B fragment was determined by digestion with BamHI, and the exons and flanking intronic DNA were sequenced to ensure that the exons and flanking intronic sequences of the cloned DNA exactly matched that of the genomic DNA. The mutation in DNA from subject 1 was contained in PCR A, and the mutation in DNA from subject 2 was contained in PCR B. Combination of PCR A from subject 2 and PCR B from an unaffected subject enabled construction of a wild-type CYP17 gene (Fig. 1Go). The constructs were used to transform Escherichia coli strain DH5{alpha} (Invitrogen), and plasmid DNAs were isolated by Wizard Plus SV Miniprep DNA Purification System (Promega, Madison, WI).

HEK-293 cell expression, activity assays, and RNA analysis

After construction, 6 µg each of each pcDNA3 vector containing the entire wild-type or mutant CYP17 genes (or empty vector) were transfected into HEK-293 cells by the calcium phosphate method (15). Thirty hours later, 0.1 µM [3H]progesterone (90,000 cpm; PerkinElmer Life Sciences, Norwalk, CT) was added in fresh medium for 17 h of incubation. The medium was then collected and submitted to extraction, chromatography, and autoradiography (15, 16). After removing the incubation medium, the cells were washed with 1 ml PBS and harvested by incubation with 10 mM disodium EDTA in PBS at room temperature for 10 min. The resulting cell suspension was pelleted by centrifugation and frozen at -70 C. RNA was extracted (Micro-Fast Track 2.0, Invitrogen) according to the manufacturer’s protocol. RT-PCR amplification of cDNAs was performed with 100 pg mRNA using the SuperScript OneStep RT-PCR system with PlatinumTaq (Invitrogen). Amplifications focused on regions surrounding the two CYP17 mutations using primers E2E4S1 and E2E4AS1 primers for subject 1 (amplification of transcripts from exons 2 through 4) and E3E5S1 and E3E5AS1 for subject 2 (spanning exons 3 through 5; Table 2Go). These amplicons were submitted for direct sequencing using the same sense primers (Table 2Go). Transfection experiments, including enzyme assay, RT-PCR, and sequencing, were repeated a second time for confirmation.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
DNA sequencing revealed that subject 1 is a compound heterozygote for Y329D (CYP17 exon 6) and substitution of CG for the AG at position g.2306 (-2) of intron 2, which is the AG of the splice acceptor site for exon 3. Subject 2 is homozygous for a deletion of four of the five T bases at g. 3187–3191 of intron 3 (-10 to -14), which lies in the pyrimidine-rich flanking intronic DNA 5' of the splice acceptor site of exon 4.

To determine whether these intronic mutations altered CYP17 mRNA splicing, the entire CYP17 genes (wild-type and mutations) were cloned into the mammalian expression vector pcDNA3 (Fig. 1Go) and transfected into HEK-293 cells. The cells were allowed to express the cloned genes, and CYP17 mRNA and CYP17 enzymatic activity were both analyzed in each experiment. The wild-type gene yielded the expected CYP17 mRNA, and this mRNA was translated into active protein, which metabolized progesterone to 17{alpha}-hydroxyprogesterone (Fig. 2Go). In contrast, cells transfected with plasmids containing the intronic mutations did not express active protein, as evidenced by the lack of progesterone (Fig. 2Go) or pregnenolone (not shown) 17{alpha}-hydroxylation.



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  		FIG. 2. Autoradiogram of progesterone metabolites from transfected HEK-293 cells. HEK-293 cells were transfected with pcDNA3 containing the wild-type CYP17 gene or the mutated genes (AG to CG or TTTT deletion). Thirty hours after the transfection, [3H]progesterone was added for 17 h, and the steroids were extracted and chromatographed. Cells expressing the wild-type CYP17 gene convert most of the progesterone to 17{alpha}-hydroxyprogesterone (17OHProg), whereas the cells expressing the AG to CG (subject 1, S1) or TTTT deletion (subject 2, S2) mutations yield only background metabolites formed by mock-transfected cells (Mock).

 
To obtain direct evidence linking the absence of 17{alpha}-hydroxylase activity with aberrant mRNA splicing, we performed RT-PCR on RNA extracted from the cells transfected with plasmids encoding the CYP17 genes. Exonic PCR primers were chosen to span the splice sites in question, and both amplifications yielded the expected products of about 450 bp with mRNA derived from cells expressing the wild-type CYP17 gene (Fig. 3Go). Direct sequencing of the RT-PCR products from these cells confirmed that introns 2 and 3 were correctly excised (Fig. 4Go). The mRNA derived from cells expressing the AG to CG mutation from subject 1 yielded an amplicon (exons 2–4) of approximately the predicted length (Fig. 3AGo), but mRNA derived from cells expressing the TTTT deletion from subject 2 yielded an amplicon (exons 3–5) that was about 90 bp shorter than predicted (Fig. 3BGo).



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  		FIG. 3. RT-PCR amplification of mRNA from HEK-293 cells expressing wild-type or mutant CYP17 genes. A, Diagram of reverse transcription (RT) of mRNA and cDNA amplification (PCR). Sequential RT and PCR in one tube was accomplished using exonic primers flanking the mutations AG to CG in intron 2 ({downarrow}; RT-PCR1) or TTTT deletion in intron 3 ({uparrow}; RT-PCR2). B, Amplification products of RT-PCR separated by agarose gel electrophoresis and ethidium bromide staining. The expected sizes of amplicons from the RT-PCR reactions are both about 450 bp (Std, standards). The size of the amplicon derived from mRNA of cells expressing mutation AG to CG (S1) is indistinguishable from that derived from cells expressing wild-type CYP17 (WT). The RT-PCR product from cells expressing the TTTT deletion mutation (S2) is 87 bp shorter than the same amplification product derived from WT cells due to excision of exon 4 during mRNA splicing.

 


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  		FIG. 4. Direct sequencing of RT-PCR products from mRNA of HEK-293 cells expressing CYP17 genes. A, Sequencing of exons 2 and 3 from RT-PCR1. The electropherograms correspond to amplicons derived from mRNA in cells expressing wild-type CYP17 (WT; top) or AG to CG mutation of subject 1 (AG to CG; bottom). Both sequences are identical to the end of exon 2 (solid vertical line), but the AG to CG mutation uses the first AG in exon 3 as the splice acceptor (dashed line), deleting the TTTGTCAG sequence from the 5' end of exon 3. B, Sequencing of exons 3–5 from RT-PCR-2. The electropherogram shows the sequence of amplicons derived from mRNA in cells expressing the TTTT deletion mutation. Exon 4 is deleted entirely, and the sequence demonstrates that exon 3 is spliced cleanly to exon 5.

 
The RT-PCR product derived from the CYP17 gene of subject 1 gave wild-type sequence up to the CG mutation, which did not splice at that site. Instead, the splicing machinery used the next closest AG, located 7–8 bp into exon 3 (Fig. 4AGo). This altered splicing deleted amino acids 146–148, shifted the reading frame, and introduced a TGA stop codon 18–20 bp downstream from the new splice site. The RT-PCR product derived from subject 2 lacked 87 bp (29 amino acids) corresponding to the entirety of exon 4 (Fig. 4BGo). This sequence indicates that the deletion of TTTT from the pyrimidine-rich intron-exon junction resulted in the excision of exon 4 during splicing (exon skipping).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Mutations in the CYP17 gene cause 17OHD, and most mutations identified in 17OHD patients occur within coding regions and change one or more amino acids in the protein product. To confirm that such missense mutations are responsible for the loss of enzyme activity, the mutation is introduced into the CYP17 cDNA by site-directed mutagenesis, and the mutant protein is expressed in a heterologous system to compare its catalytic activity to that of wild-type CYP17. Well described and reliable methods exist to construct mutant CYP17 cDNAs and to assay these proteins containing amino acid substitution mutations. Experimental demonstration of the functional consequences from intronic mutations, however, is more problematic, because mutations in noncoding regions do not necessarily interfere with protein expression and enzyme activity.

Intronic mutations found in patients with 17OHD lay near intron-exon boundaries, and these mutations appear to disrupt pre-mRNA splicing. An analysis of 400 vertebrate genes (17) has yielded consensus sequences that are essential for correct splicing (Fig. 5Go). The 10 bp (-4 to +6, including the GT at the 5' start of the intron) constitute the splice donor site, and a G at position +5 is highly conserved (18). Nucleotides -14 to +1 (including the AG at the 3' end of the intron) comprise the splice acceptor site (19). Splice acceptor sites are pyrimidine-rich, a property that appears to be necessary for intron recognition by the RNA/protein spliceosome complex (20, 21), leading to the sequential phosphotransesterifications that appear to constitute the splicing process (22).



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  		FIG. 5. Diagram of mRNA splicing of the wild-type CYP17 gene and splice acceptor site mutations. A, The dinucleotide AG (underlined, top sequence) is the alternative splice acceptor site that alters the exon 3 reading frame and introduces the stop codon TGA (underlined, bottom sequence). The substitution A to C in intron 2 is shown in the shaded box (top sequence), and the G following the alternate AG is highlighted by the arrow ({uparrow}) in all three sequences. B, The 3' end of intron 3 contains the pyrimidine-rich (y) consensus sequence of splice acceptor sites, including the sequence ttttt (underlined), four of which are deleted in subject 2 (t), causing excision of exon 3 during mRNA splicing.

 
To date, two CYP17 mutations in splice donor sites have been described in three Japanese patients with the classical form of 17OHD: 1) a homozygote for G->A (+5) in intron 7 (14), 2) a compound heterozygote for G->T (+5) in intron 2 and Trp17Stop (12), and 3) a compound heterozygote for the same G->T (+5) in intron 2 and His373Leu (13). In case 2, testicular mRNA contained a mixture of correctly spliced transcripts and transcripts missing exon 2 (exon skipping). This result suggests that mutation G->T (+5) in intron 2 destroyed the splice donor site, although this interpretation is complicated by the existence of a wild-type allele in this compound heterozygote. Evidence for aberrant mRNA splicing in cases 1 and 3 derive from minigene experiments in which two exons and the intervening intron, mutant or wild type, are transcribed using a mammalian expression plasmid. Although minigene experiments are somewhat artificial, both mutations occurred at the highly conserved G at position +5 of the splice donor site, and similar mutations at analogous G (+5) positions in other genes have been shown to cause other genetic diseases, including hemophilias A and B (17). Thus, there is no reason to doubt that these mutations severely disrupt pre-mRNA splicing.

Herein, we describe the first two mutations in CYP17 splice acceptor sites that are associated with 17OHD. The AG to CG mutation in intron 2, which destroys the AG motif that is critical for the splicing process, is likely to completely disrupt RNA splicing. On the other hand, it is not so obvious that the TTTT deletion in intron 3 should yield no correctly spliced transcripts that can be translated into active protein. Although minigene experiments may demonstrate that exon skipping occurs, it is difficult to determine whether any normally spliced transcripts are produced because the shorter (abnormal) transcripts amplify more efficiently during RT-PCR. Furthermore, no functional protein product is produced in either case, limiting conclusions from such experiments. In an effort to design a more robust and generally applicable method to analyze both the mRNA transcripts and protein products derived from CYP17 genes with intronic mutations, we subcloned the entire CYP17 genes (wild type and mutations) into the pcDNA3 expression vector and transfected these 11.5-kb plasmids into HEK-293 cells. This experimental strategy, which has been successfully employed to isolated primate CYP17 cDNAs from genomic DNA (23), allowed us to determine not only the sequence of the mRNA species produced, but also the net enzyme activity resultant from the expression of these genes.

Although 17{alpha}-hydroxylase activity and correctly spliced transcripts were easily demonstrated for cells transfected with the wild-type CYP17 gene, sensitive radiochemical assays failed to detect any enzyme activity in cells transfected with the mutant genes. Direct sequencing of the mRNA transcripts confirmed that aberrant splicing had occurred for both mutations. The AG to CG mutation in intron 2 diverted splicing to a cryptic splice acceptor (AG) site 7–8 bp downstream in exon 2, which shifted the reading frame and generated a premature stop codon 8 residues into exon 2. The TTTT deletion in intron 3, which shortens the pyrimidine-rich stretch in that splice acceptor site, resulted in the splicing of exon 3 to exon 5, skipping exon 4 entirely. In addition, we could only amplify the full-length CYP17 mRNA from cells transfected with the wild-type gene (not shown), so primers were designed to amplify smaller pieces of the cDNAs to isolate abnormally spliced transcripts. This result suggests that CYP17 mRNA splicing and/or stability were more globally impaired for the mutations, an observation that would not have been possible with data from minigene experiments alone.

Subject 1 is a 46,XX compound heterozygote for the intron 2 splice mutation and Y329D in exon 6. Her clinical picture was compatible with partial 17OHD: attainment of complete sexual development in the presence of mineralocorticoid excess and hypertension. Chronically elevated gonadotropins, particularly FSH, in the presence of a partial block in androgen synthesis and anovulation, stimulated the growth of large, hemorrhagic cysts in both ovaries. We could not demonstrate any correctly spliced transcripts from the allele with the intronic mutation, which is consistent with the critical role of the AG dinucleotide motif at 3' end of the intron for proper splicing (17). We attribute the development of secondary sexual characteristics and regular menses to the other allele with mutation Y329D, as the Y329D mutation retains about 5% of wild-type 17{alpha}-hydroxylase activity (11), which is consistent with partial 17OHD (24). Subject 2, in contrast, was homozygous for the TTTT deletion in intron 2, and this 46,XY individual had severe hypertension, hypokalemia, and sexual infantilism consistent with complete 17OHD.

In summary, transfection and expression of the entire CYP17 gene, instead of the affected intron and flanking exons only, is a feasible approach for functional studies of intronic CYP17 mutations. This approach has the advantage of enabling the analysis not only of the mRNA transcript(s), but of the protein products as well, and this design may be preferable to minigene experiments in cases where mRNA splicing may be only partially disrupted. The AG to CG substitution in intron 2 and the TTTT deletion in intron 3 are the first two CYP17 gene mutations reported in splice acceptor sites in which 17OHD has been shown to result from aberrant RNA splicing.


   Acknowledgments
 
We thank Kerri Kwist for assistance with the experiments, and Dr. Jean Wilson for clinical data and fibroblasts from subject 2.


   Footnotes
 
This work was supported by NIH grants K08-DK-02387 and R03-DK-56641 (to R.J.A.) and by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (96/7449-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (0690/00-7; to C.E.K.).

Results of this work were presented in abstract form at the 84th Annual Meeting of The Endocrine Society, San Francisco, CA, June 2002.

Abbreviation: 17OHD, 17-Hydroxylase deficiency.

Received June 12, 2003.

Accepted September 11, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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