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Experimental Studies |
-Hydroxylase Gene in 17-Hydroxylase Deficiency
Third Department of Internal Medicine, Miyazaki Medical College (H.Y., M.N., M.M., S.M.), Kiyotake, Miyazaki 889–16; and the National Cardiovascular Center Research Institute (M.M., K.K.), Suita, Osaka 565, Japan
Address all correspondence and requests for reprints to: Masamitsu Nakazato, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889–16, Japan. E-mail: nakazato{at}post.miyazaki-med.ac.jp
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Abstract |
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-Hydroxylase deficiency (17OHD) is an autosomal recessive disorder
that produces an excess of mineralocorticoids and sexual
differentiation abnormalities. Using DNA sequencing analysis of the
17-hydroxylase (CYP17) gene from a Japanese patient
with 17OHD, we identified a new type of genetic abnormality in this
disease, a G to A transition at position +5 in the splice donor site of
intron 7 of the CYP17 gene. In vitro
expression analysis of an allelic minigene that consists of exons 6–8
of the patient’s CYP17 gene showed that the transition
causes the skipping of exon 7. This exon skipping alters the
translational reading frame of exon 8 and introduces a premature stop
codon (TAA) at amino acid position 410 proximal to the heme
iron-binding site essential for the enzymatic activity of
CYP17. Restriction enzyme analysis showed that the
patient is homozygous for the mutated CYP17 gene, and
the parents are heterozygotes. This is the first reported patient with
17OHD caused by the splice site mutation in the CYP17
gene. |
Introduction |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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-hydroxylase (P450c17) is an important component of the
steroidogenic synthesis pathway that leads to the production of
17-hydroxysteroids and adrenal and gonadal sex steroids. The human
17-hydroxylase (CYP17) gene, a single copy gene located
on chromosome 10q24.3 (1, 2), consists of 8 exons that span 6569 bases
and encode a protein of 508 amino acids (3). This gene is expressed in
the adrenal cortex and gonads (4). P450c17 catalyzes both the
17-hydroxylation of pregnenolone or progesterone and the 17,20-lyase
reaction of 17-hydroxylated pregnenolone to produce the
C19 steroid precursors of androgens and estrogens.
17-Hydroxylase deficiency (17OHD), an autosomal recessive disorder,
is relatively rare; 130 patients have been reported (Refs. 5–8 and
references therein). Genetic abnormalities in the CYP17 gene
affect both adrenal and gonadal steroidogenesis. Impaired production of
cortisol and sex steroids induces the elevation of plasma ACTH and the
overproduction of mineralocorticoids, resulting in hypertension,
hypokalemia, and bilateral adrenal hyperplasia. In addition, the
impaired production of sex steroids leads to abnormal sexual
development.
To date, 20 deleterious mutations have been reported in this disorder (5, 6, 7, 8). These mutations occur in 7 exons of the gene, excluding exon 7, and consist of missense and nonsense mutations as well as small insertions or deletions that alter the reading frame of the gene.
In the transcription step in eukaryotic genes, they first produce premessenger ribonucleic acids (pre-mRNAs) that carry intervening noncoding sequences (introns), then the introns are removed by splicing before the mRNA is transported to the ribosomes where it is translated to protein. The splicing process requires consensus nucleotide sequences at the 5'- and 3'-splice sites of the pre-mRNAs. Mutations at the splice junction sequences cause human genetic diseases by altering the accuracy of mRNA splicing (9). We here report a new type of CYP17 gene mutation, a G to A transition at position +5 in the splice donor site of intron 7, in a Japanese patient with 17OHD.
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Subject and Methods |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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The patient, a 32-yr-old Japanese phenotypic woman, has been
reported in detail by Yazaki et al. (10). In summary, her
clinical manifestations were generalized muscle weakness due to
hypokalemic myopathy, primary amenorrhea, and lack of pubertal
development. Laparoscopy showed intraabdominal testes and no uterus.
Her karyotype was 46,XY. She is the offspring of a first cousin
marriage. Her parents showed hypertension, but no abnormalities in
sexual development. Basal plasma hormone levels are summarized in Table 1
. Glucocorticoid replacement therapy normalized the
plasma levels of ACTH, 17-deoxysteroids, and potassium.
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Genomic DNA was extracted from the patient’s peripheral blood leukocytes. Eight exons of the CYP17 gene, including the exon-intron boundaries, were amplified by PCR with a GeneAmp PCR reagent kit (Perkin-Elmer/Cetus, Norwalk, CT). The oligonucleotide primers and PCR conditions were described by Monno et al. (11). The PCR products were ligated with pT7Blue T-Vector (Novagen, Madison, WI), then transformed into Escherichia coli XL1-Blue supercompetent cells (Stratagene, La Jolla, CA). Ten colonies were selected and cultured, then purified with Qiagen resin (Qiagen, Chatsworth, CA). The plasmid DNA was sequenced in an ABI 373A automated sequencer (Applied Biosystems, Foster City, CA).
Determination of zygosity
A 215-bp fragment of the CYP17 gene that included a G
to A transition at position +5 in the splice donor site of intron 7 was
amplified with a subcloning primer set (Fig. 2A). PCR conditions were a
hot start with 0.5 U AmpliTaq, followed by 30 cycles of denaturation at
94 C for 50 s, annealing at 62 C for 50 s, and extension at
72 C for 100 s. The PCR product was digested at 37 C for 2 h
with 2.5 U of the restriction enzyme TfiI (New England
Biolabs, Beverly, MA), then electrophoresed on a 3% agarose gel
(NuSieve GTG Agarose, TaKaRa Shuzou Co., Shiga, Japan).
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Exons 6–8 of the patient’s mutant CYP17 gene and
those of the normal CYP17 gene from a control subject were
amplified using the Expand Long Template PCR System (Boehringer
Mannheim Biochemica, Philadelphia, PA). The respective PCR products,
consisting of 2057 bp, were ligated to EcoRI/NotI
adaptors and size-fractionated in a Sepharose CL-4B column (Pharmacia
Biotech, Piscataway, NJ). The resulting products were ligated to
pBluescript vector and cloned. Ligation of the products to the
EcoRI/NotI adaptors was confirmed by direct DNA
sequencing. The inserts were cleaved from the plasmid DNA with the
restriction enzyme NotI (Toyobo Biochemicals, Osaka, Japan),
then ligated to the expression vector pcDNAI/Amp (Invitrogen, San
Diego, CA; Fig. 3). The chimeric plasmids were cloned, then purified
with Qiagen resin. Two micrograms of the mutant or normal chimeric
plasmid were transfected into COS-7 cells by the lipofectamine method.
The transfected cells were plated in six-well plastic culture dishes
and incubated at 37 C for 6 h in serum- and antibiotic-free DMEM.
The medium then was changed to one containing 10% FCS and antibiotics
(100 IU/mL penicillin and 100 µg/mL streptomycin) and again changed
24 h later. After 48 h, the transfected cells were washed
once with phosphate-buffered saline, then digested with trypsin and
collected. mRNA was extracted from the cells with a Micro-FastTrack
mRNA Isolation Kit (Invitrogen). The first strand complementary DNA
(cDNA) was synthesized with 25 ng mRNA sample, 110 U ribonuclease
inhibitor, 6 µmol/L MgCl2, deoxy-NTP mixture (8 mmol/L
each), 1 x PCR buffer (GeneAmp PCR Reagent Kit,
Perkin-Elmer/Cetus), 7.5 µmol/L oligo(deoxythymdine)18
primer, and 200 U Superscript RT ribonuclease H- reverse
transcriptase (Life Technologies, Gaithersburg, MD). Reverse
transcription was performed at 42 C for 30 min, after which the mixture
was incubated at 94 C for 3 min to inactivate the reverse
transcriptase. The resulting cDNA was subjected to PCR amplification
with 2 µmol/L primers (sense: 5'-AAGCTCTACGAGGAGATTGACC-3',
6430–6451 bp; antisense: 5'-TGTGTTGTGGGGCCACATAG-3', 8422–8441 bp)
and 2.5 U Taq DNA polymerase (Fig. 3). PCR conditions were
32 cycles of denaturation at 94 C for 30 s, annealing at 55 C for
30 s, and extension at 72 C for 1 min. PCR products of 420–612 bp
were excised from the 2% agarose gel (FMC BioProducts, Rockland, ME)
and purified with Qiagen resin. Portions of the purified PCR products
were further amplified with an inner oligonucleotide primer set (sense:
5'-CTATCAGTGACCGTAACCGTCT-3', 6479–6500 bp; antisense:
5'-CAGGATCTCACCTATACAGGAG-3', 8186–8207 bp; Fig. 3). The products of
this nested PCR were electrophoresed on a 2% agarose gel, purified
with Qiagen resin, then sequenced in an ABI 373A automated
sequencer.
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Results |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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The entire coding region of the patient’s CYP17 gene,
including the exon-intron boundaries, was sequenced. The patient had a
G to A transition at position +5 in the splice donor site of intron 7
(Fig. 1). All 10 clones of exon 7 examined here had this
mutation. No base changes were found in the 8 exons or other
exon-intron boundaries.
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The PCR product from the mutant allele with a G to A transition at
+5 position in intron 7 was cut into 134- and 81-bp fragments by
TfiI digestion, but wild-type allele was undigested (Fig. 2A
). The patient gave only 134- and 81-bp fragments on
TfiI digestion (Fig. 2B), indicative that she was homozygous
for the mutated CYP17 gene. Both of her healthy parents had
the normal 215-bp fragment in addition to the abnormal 134- and 81-bp
fragments, indicating that they were heterozygous carriers. Fifty
normal subjects had only the 215-bp fragment.
Expression of the CYP17 minigene in COS-7 cells
The patient’s CYP17 mRNA needed to be analyzed to
determine whether the G to A transition causes abnormal transcription
of the CYP17 gene, but this was impossible because the
CYP17 gene is expressed only in the adrenal gland of the
patient whose intraabdominal testes had already been extirpated. We,
therefore, made an in vitro expression study using the
chimeric minigene system to analyze the transcription of the patient’s
CYP17 gene. We first constructed two chimeric minigenes that
consisted of an expression vector pcDNAI/Amp and the mutant or
wild-type CYP17 gene, which spans exons 6–8 (Fig. 3). After the respective minigenes were expressed in
COS-7 cells, the splice site selection patterns for exons 6–8 were
assessed from the cDNA sizes that corresponded to the expressed mRNAs.
The mRNA first was subjected to the reverse transcription-PCR (RT-PCR),
then to nested PCR because there was too little RT-PCR product to
analyze by electrophoresis. The nested PCR product of the chimeric
minigene that included the wild-type CYP17 gene was 314 bp
(Fig. 4A) as expected. The transcript of the mutant-type
minigene was 210 bp, 104 bp shorter than that of the wild-type product.
The deletion corresponded to exon 7 with a size of 104 bp. We confirmed
these two 314- and 210-bp products by DNA sequencing analyses. The
former consisted of exons 6, 7, and 8, whereas the latter consisted
only of exons 6 and 8 (Fig. 4B). This exon skipping altered the reading
frame of exon 8, resulting in a premature stop codon (TAA) 90 bp
downstream from the exon 6–8 boundary (codon 380; Fig. 4C).
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Discussion |
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TopAbstractIntroductionSubject and MethodsResultsDiscussionReferences |
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-hydroxysteroids and sex steroids, hypertension due to the high
plasma levels of mineralocorticoids, and bilateral adrenal hyperplasia
due to oversecretion of ACTH. Twenty different mutations of the
CYP17 gene have been reported to date in 17-hydroxylase
and 17,20-lyase deficiency syndromes (Refs. 5–8 and references
therein). These mutations include small base pair duplications (7-bp
duplication in exon 2, Ile duplication in exon 2, and 4-bp duplication
in exon 8), small base pair deletions (Phe in exon 1, 2-bp deletion in
exon 5, 1-bp deletion in exon 8, and Asp-Ser-Phe deletion in exon 8),
518-bp deletion with a 469-bp insertion in exons 2–3, missense
mutations (Tyr64Ser, Gly90Asp,
Arg96Trp, Ser106Pro, Pro342Thr,
His373Leu, Arg440His, and
Arg496Cys), and nonsense mutations (Trp17End,
Glu194End, Arg239End, and
Gln461End). Four of the patients studied had compound
heterozygous mutations: Glu194End and
Arg239End, Tyr64Ser and duplication of Ile in
exon 2, Arg239End and Pro342Thr, and
Gln461End and Arg496Cys. The others were
homozygous for their individual mutations. RNA splicing occurs on the spliceosome, a complex assembly of small nuclear ribonucleoprotein particles composed of a variety of small nuclear RNAs and associated proteins. The nucleotide sequence of pre-mRNA and its secondary structure are necessary elements for recognition of the splice donor and splice acceptor sites as well as for cleavage of the intron by the spliceosome. U1 small nuclear ribonucleoprotein particles binds directly to the 5'-splice donor site, a process mediated by base-pairing between the 5'-end of the U1 snRNA and the complementary nine nucleotides at the mRNA splice junction (12). Most introns begin with GT and end with AG in the nuclear genes of eukaryotes (GT-AG rule). The consensus DNA sequence of the 5'-splice donor site in vertebrate genes is GTAGAGT. Various mutations in the vicinity of mRNA splice junctions have been shown to cause abnormal splicing, resulting in exon skipping or, less frequently, creation of novel cryptic splice sites (9). The guanine at position +5 in the splice donor site is highly conserved in vertebrate genes (13), and the G to A transition at this position is responsible for hemophilia A, hemophilia B, ß-thalassemia, osteogenesis imperfecta II, Lesch-Nyhan syndrome, acatalassemia, protein C deficiency, and protein S deficiency (Ref. 9 and references therein). The single base change at the +5 position in these hereditary disorders causes skipping of the preceding exon, which produces a truncated protein with no biological activity. Replacement of guanine with another nucleotide at position +5 is predicted to reduce the stability of the base-pairing of the 5'-splice site with the complementary region of U1 small nuclear RNA (9).
Using an in vitro expression study with the chimeric minigene system, we verified that the G to A transition in intron 7 of the patient’s CYP17 gene caused the skipping of exon 7. This skipping altered the reading frame downstream from codon 380, which created a premature termination signal at codon 410. The codons between 435 and 455 of the CYP17 gene are well conserved in many species, in particular Cys442, the ligand of the catalytic heme iron of the cytochrome P450 enzyme, which is essential for enzyme activity (14). Three nonsense mutations (Trp17End, Glu194End, and Arg239End) and three reading frame shifts (7-bp duplication in exon 2, 2-bp deletion in exon 5, and 518-bp deletion with a 469-bp insertion in exons 2–3) produced premature stop codons proximal to Cys442. These mutations completely abolished the enzymatic activity of P450c17 in in vitro expression studies that used truncated P450c17 molecules that lack Cys442 (5). The splice junction mutation found in intron 7 in our study, therefore, is considered to be null for P450c17 activity.
Because of parent consanguinity and the known autosomal recessive inheritance of this disease, the patient’s genotype was considered to be homozygous for the mutation. This was confirmed by restriction enzyme analysis and by the heterozygosity found for her parents.
We have described a novel genetic mutation of the CYP17 gene. The knowledge gained from this study provides new insight for better understanding of the molecular pathogenesis of 17OHD.
Received October 9, 1996.
Revised March 4, 1997.
Accepted March 12, 1997.
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References |
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, unaffected; 
and ,
carriers; =, consanguineous marriage; NE, not examined.
