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Original Studies |
Department of Public Health and Cell Biology, Section of Anatomy (I.S., P.G., P.R., R.G.), University of Tor Vergata, 00133 Rome; Research and Cure Scientific Institute, Bambino Gesù, Unit of Auxology (M.C.), 00050 Palidoro; and Department of Internal Medicine, Section of Endocrinology (C.M., G.F.), University of Tor Vergata, 00133 Rome, Italy
Address all correspondence and requests for reprints to: Prof. Geremia Raffaele, Dipartimento di Sanita Pubblica e Biologia Cellulare, Sezione di Anatomia Umana, Via di Tor Vergata, 00133 Rome, Italy. E-mail: geremia{at}uniroma2.it
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Abstract |
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In this study we present an 11-yr-old XY girl, with clinical manifestations peculiar for impaired androgen biological action, including female phenotype, blind-ending vagina, small degree of posterior labial fusion, and absence of uterus, fallopian tubes, and ovaries. At the time of the diagnosis the patient had a FSH/LH ratio according to the puberal stage, undetectable 17ß-estradiol, and high levels of testosterone (80.1 ng/mL). After bilateral gonadectomy, performed at the age of 11 yr, histological examination showed small embryonic seminiferous tubules containing prevalently Sertoli cells and occasional spermatogonia together with abundant fibrous tissue. Molecular study of the patient showed a guanine to thymine transversion in position +5 of the donor splice site in the junction between exon 6 and intron 6 of the AR gene. The result of RT-PCR amplification of the AR messenger ribonucleic acid from cultured genital skin fibroblasts of the patient suggests that splicing is defective, and intron 6 is retained in most of the receptor messenger ribonucleic acid molecules. We show by immunoblotting that most of the expressed protein lacks part of the C-terminal hormone-binding domain, and a small amount of normal receptor is observed. This is probably responsible for the reduced binding capacity in genital skin fibroblasts of the patient.
The molecular basis of the alteration in this case is a novel, uncommon mutation, leading to a phenotype indicative of a partial androgen insensitivity syndrome, Quigley’s grade 5.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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After AR gene cloning (15), it was possible to study the molecular defects of AR in human androgen insensitivity syndrome (AIS). Mutations of the AR are a frequent cause of AIS, a polymorphic disease with a broad range of phenotypes (16), ranging from the complete form (CAIS), with female external genitalia, blind-ending vaginal pouch at birth, and gynecomastia at puberty, through partial forms (PAIS), characterized by ambiguous genitalia or mild hypospadias, to milder forms of AIS, in which the phenotype is male, but there is azoospermia and infertility (4). At the molecular level, most of the alterations found in the AR gene are point mutations, and the majority of changes reported to date are located in the ligand-binding domain.
In the subject described in this report, a point mutation affecting the splice region at the junction between exon 6 and intron 6 is present. This mutation is a transversion that changes a guanine to a thymine at position +5 of the donor splice site, which is known to be important for correct splicing. We present data indicating that the presence of this mutation modified ribonucleic acid (RNA) splicing, with a larger messenger RNA (mRNA) as a result of persistence of intron 6 in the mature transcript. As a consequence, a stop codon within the unspliced intron sequence causes the expression of a truncated protein lacking part of the ligand-binding domain. The PAIS phenotype of the patient is probably due to a small amount of receptor correctly spliced and expressed.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The patient was a female, first referred at 8 yr and 11 months
of age for bilateral and inguinal hernia. She was a product of
full-term uncomplicated pregnancy. At birth, weight was 3.2 kg, and
length was 50 cm. Normal milestones were recorded during infancy. At
the first clinical observation she was 136.7 cm tall (+1.11
SD score according to Tanner female standard curves) and
weighed 30 kg (-10%), and Tanner stage was P1B1. General physical
examination, intellectual function, and muscle power were normal.
Normal female external genitalia with slight clitoromegalia were found.
Bilateral inguinal hernias were evident. In contrast with the complete
female phenotype, the karyotype was 46,XY. Pelvic computed tomography
and ultrasonography localized bilateral intraabdominal structures
contiguous to the posterior surface of the bladder. The endocrine
biochemical pattern demonstrated a FSH/LH ratio according to the
pubertal stage, normal 17
-hydroxyprogesterone (1.1 ng/mL),
undetectable 17ß-estradiol, and high levels of testosterone (80.1
ng/mL). Thus, the diagnosis of PAIS with female phenotype was based on
both clinical (Tanner stage P1B1, female external genitalia with 2-cm
deep blind-ending vagina, mild clitoromegaly and small degree of
posterior labial fusion, and lack of Mullerian structures by
ultrasonography) and karyotypic evidence. At the age of 11 yr, the
patient underwent bilateral gonadectomy. Bilateral testes, 1.6 and 2.2
cm in diameter, were localized and removed. They had a thickened
albuginea membrane. The histological examination showed small embryonic
seminiferous tubules without lumen and with thickened basal membrane.
Tubules contained prevalently Sertoli cells and occasionally
spermatogonia and were joined with abundant fibrous tissue containing
bundled spindle-shaped cells, probably related to Leydig precursor
cells. Tumor-like nodules (Sertoli cells amartomas) were also found.
Surgical specimens contained some smooth muscle tissue histologically,
consistent with an incompletely developed uterine body. A genital skin
biopsy was performed during surgery. All studies of this patient were
performed after obtaining the parent’s written informed consent and
were approved by the ethical committee of the University of Rome, Tor
Vergata.
Cell cultures
Genital skin fibroblasts obtained from patient and control skin biopsy were grown to confluence in 10-cm dishes in DMEM (Life Technologies, Inc., Gaithersburg, MD; catalogue no. 61965–026), supplemented with 10% bovine calf serum, nonessential amino acids (Life Technologies, Inc., catalogue no. 11140–035), 100 U/mL penicillin-streptomycin, and 10 µg/mL gentamicin in a 5% CO2 atmosphere at 37 C.
AR binding assay
Androgen binding was investigated with a whole cell assay, using genital skin fibroblasts, cultured as previously described, in multiwell dishes (Falcon, catalogue no. 353046; Becton Dickinson Labware, Franklin Lakes, NJ). Confluent cells were incubated 2 h at 37 C with binding medium (serum-free DMEM medium, containing 0.1% BSA) and subsequently incubated in duplicate at 37 C for 2 h using binding medium containing increasing concentrations (from 0.03 to 2.5 nmol/L) of methyltrienolone ([3H]R1881; SA, 85 Ci/mm; NEN Life Science Products, Boston, MA; catalogue no. NET 590), in the presence or absence of 1 mmol/L unlabeled ligand.
Cells were then washed twice with phosphate-buffered saline (PBS) containing 0.1% BSA and 20 mmol/L Tris, pH 7.4, and once with PBS only. The cells were trypsinized, resuspended in saline containing BSA and Tris, centrifuged at 1500 rpm for 10 min, washed with PBS, and centrifuged again. All operations were performed at 4 C. Supernatants were discarded, and cell pellets were resuspended in 1 mL 0.5 N NaOH (30 min, 56 C). Five hundred microliters were taken for liquid scintillation counting, and 200 µL were used for protein determination as previously described (17). Scatchard calculations were performed using Prism software (version 2.01, GraphPad Software, Inc., San Diego, CA).
DNA, RNA, and protein extraction
Genomic DNA was extracted from peripheral whole blood of the patient and normal controls using the DNA isolation kit for mammalian blood (Roche Molecular Biochemicals, Indianapolis, IN; catalogue no. 1667327) and following the manufacturer’s instructions.
Total RNA was extracted from cultures of genital skin fibroblasts of the patient and from prostatic tissue and genital skin fibroblasts of controls using TriPure isolation reagent (Roche Molecular Biochemicals, catalogue no. 1667165), following the manufacturer’s instructions.
Proteins were extracted from cultured genital skin fibroblasts and fragments of testis of the patient and from two different prostate tissues of subjects with benign hypertrophy. Cultured cells were scraped, and tissue fragments were homogenized in 20 mmol/L HEPES (pH 7.5), 120 mmol/L KCl, 0.1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 mmol/L ß-glycerophosphate, 10 µg/mL leupeptin, and 10 µg/mL aprotinin. The extracts were centrifuged for 15 min at 12,000 x g at 4 C.
Amplification of AR exons by PCR
Exons of human AR were amplified selecting 12 pairs of primers according to Lubhan (15). The oligonucleotides were synthesized on a DNA synthesizer (model 391, from PE Applied Biosystems, Foster City, CA). Amplification by the PCR took place in 50-µL reaction mixtures containing about 25 ng genomic DNA. PCR mixture contained 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L of each deoxy (d)-NTP, 0.25 U Thermus aquaticus (Taq) DNA polymerase (Roche Molecular Biochemicals, catalogue no. 1418432), and 1 µmol/L of each oligonucleotide. Amplification was performed with an initial denaturation step of 5 min at 95 C, followed by 35 cycles; each cycle included denaturation for 1 min at 95 C, primer annealing for 1 min at 60 C, and primer extension for 1.5 min at 72 C. A final passage of extension of 10 min at 72 C was performed. The reaction was controlled by loading 10 µL reaction mixture on a 1% agarose gel.
Single strand conformational polymorphism (SSCP)
Point mutations in the AR gene were analyzed by SSCP.
[-32P]dATP (2 µCi; Amersham Pharmacia Biotech, Arlington Heights, IL; catalogue no. PB
10204) was added to each PCR reaction tube. After checking the reaction
on a 1% agarose gel, 2 µL of the reaction were diluted in 30 µL
stop mix (95% formamide, 20 mmol/L ethylenediamine tetraacetate,
0.05% bromophenol blue, and 0.05% xylene cyanol). DNA fragments were
denatured by heating at 95 C for 5 min, and 3 µL were loaded on a
nondenaturing 6% polyacrylamide gel 55 cm long. Two runs were
performed for each sample: at room temperature with 10% glycerol in
the gel or at 4 C without glycerol. The samples were run overnight,
using the Macrophor sequencing system (Amersham Pharmacia Biotech), in 1 x Tris-borate buffer at a constant voltage
of 5.5 V/cm. Autoradiography of the dried gel was carried out for
24 h with an intensifying screen.
DNA sequencing
The sequencing reactions were performed using the T7
Sequenase 2.0 PCR product sequencing kit (Amersham Pharmacia Biotech, catalogue no. US70170). The reactions were performed
according to the manufacturer’s instructions, using the same primers
of PCR amplification, and [-35S] dATP
(Amersham Pharmacia Biotech, catalogue no. AG1000A250) in
the labeling step. DNA fragments were run on a denaturing 6%
polyacrylamide gel, using the Macrophor sequencing system, in 1 x
Tris-borate buffer at a constant voltage of 27 V/cm. Autoradiography of
the dried gel was carried out for 2 days. (The sequence of exon 6 was
performed twice, using two different PCR reactions.)
RT-PCR
The RT-PCR of samples from the patient and the controls was performed using the RT-PCR kit (Stratagene, La Jolla, CA; catalogue no. 200420), according to the manufacturer’s instructions. To synthesize complementary DNA (cDNA), 5 µg total RNA were used, and the amplification reaction was performed as usual, using two primers, NF1 and NG2, that spanned from exon 6 to exon 7, including intron 6, and two primers, E3 and F3, that spanned from exon 5 to exon 6, including intron 5. The sequences of the primers used were: NF1, 5'-GGCTGCAGATGTACCGCATGCACAAGTCCCG-3'; NG2, 5'-GGCTGCAGAGGCTGCACGGAGTCCAGGA-3'; E3, 5'-GCTTCCGCAACTTACACGTGGACG-3'; and F3, 5'-TAATGCTGAAGAGTAGCAGTGCTT-3'.
Western blot
For detection of normal and mutant AR, the extracts from tissues and cell cultures (60 µg whole cell lysate) were separated on 7.5% SDS-PAGE gel, transferred onto nitrocellulose membrane (Amersham Pharmacia Biotech), and subjected to Western blot analysis as previously described (18). Briefly, first antibody incubation was carried out with 1:200 dilution of a polyclonal antibody anti-AR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; catalogue no. sc-816), second antibody incubation was carried out with a 1:2000 dilution of antirabbit IgGs antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech, included in the ECL kit). The anti-AR antibody was produced against a peptide of the NH2-terminal region of AR. According to the manufacturer’s indication, this antibody recognizes an AR band of 132 kDa from human tissues. The blot was developed using the ECL Western blotting chemiluminescence detection system (Amersham Pharmacia Biotech, catalogue no. RPN 2108). We used the program Molecular Analyst from Bio-Rad Laboratories, Inc. (version 1.4, Hercules, CA), a Windows software program for image analysis, to calculate the molecular weight of an unknown band compared to that of a known molecular standard.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The structural integrity of the AR gene-coding region was analyzed
by PCR amplification of individual exons using oligonucleotide pairs
designed at the intron-exon junctions, thus generating products
spanning the entire exons. In the case of exon 1, due to its length,
five pairs of overlapping oligonucleotides were used, whose sequences
were previously reported (15). Each amplified fragment had the correct
base pair length and agreed with the size produced using genomic DNA
from normal controls (data not shown). The fragments amplified by PCR
were screened by SSCP, a technique used for rapid identification of
point mutations (19). Mobility shift was observed only in exon 6,
suggesting a sequence alteration in this fragment. Figure 1
shows the mobility pattern at room
temperature. The same pattern was observed at 4 C (data not shown).
Sequence analysis of exon 6 of the AR gene amplified from patient’s
DNA was performed. A G
T transversion was found at nucleotide 3482 in
the splicing region, three bases downstream from the canonical donor
splice site of intron 6. The sequence was performed twice, from two
different amplified DNA, and each time by sequencing both strands.
Figure 2 shows the sequence of the
splicing region in which the point mutation was identified.
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To establish whether an altered RNA maturation could be the
consequence of this mutation, RT-PCR was carried out using primers
designed to enclose exons 6 and 7, and the intron 6. The lengths of
these fragments, as reported from Quigley (20), are 131 bp for exon 6,
158 bp for exon 7, and 800 bp for intron 6. Figure 3 shows that the amplified fragment from
control RNA measured the expected 289 bp (exon 6 plus exon 7), whereas
in that from the patient, a main band was about 1100 bp. This band is
in agreement with the expected length, including exons 6 and 7 and
intron 6, of 1089 bp, as confirmed by direct dideoxysequencing of the
RT-PCR product (data not shown). The results suggest normal RNA
maturation with intronic excision in the control and essentially lack
of splicing in the patient. The constant presence of a faint band of
the correct size in the DNA of the patient suggests that a small
proportion of the transcript is normally matured. Additional evidence
that the mutation studied affected only the splicing of intron 6,
without disturbing the correct splicing upstream, was obtained by
performing RT-PCR using two primers spanning from exon 5 to exon 6 and
including intron 5. The reported length is 145 bp for exon 5, 131 bp
for exon 6, and 4.8 kbp for intron 5 (20). Figure 4 shows that a band of 276 bp of the same
intensity is amplified from both control and patient DNA, in agreement
with a correct splicing of intron 5. Furthermore, this result confirms
that there is no skipping of exon 6.
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shows that a 132-kDa band is present in
extracts from human prostatic tissue of normal males. This band is
completely absent in extracts from testis tissue of the patient,
whereas in extracts from cultured genital skin fibroblasts, a smaller,
122-kDa band is present, together with a faint band of the right
molecular mass. These data suggest the expression of a truncated AR
and, in agreement with the results of RT-PCR experiments, of a small
amount of AR of the correct size. As for the AR molecular mass observed
in our experiments, even though the AR open reading frame encodes for a
110-kDa polypeptide, the 132-kDa band shown in the Western blot was
specific, as demonstrated by competition experiments with the
immunogenic peptide (data not shown), and it was the expected size
according to the manufacturer.
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shows almost a
3-fold reduction of the binding capacity (Bmax)
in the patient’s genital skin fibroblasts, whereas the
Kd is essentially unchanged.
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122 kDa). This band corresponds to a
truncated receptor encoded by the sequence from the ATG until the stop
codon inside intron 6, but missing exons 7 and 8. Figure 6 shows the intronic sequence with the
identified stop codon.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Here we describe a case of a 46,XY child, phenotypically female, carrying a mutation of the AR gene in the region of the splice donor site at the boundary between exon 6 and intron 6. The mutation is a transversion that changes the guanine at position +5 of the splice donor site in a thymine and almost completely abolishes the splicing of RNA. The identification of the point mutation was reached after PCR amplification, SSCP analysis, and sequencing. The patient, referred to our clinical observation for bilateral inguinal hernias, was diagnosed for AIS at the age of 8 yr. The diagnostic evaluation demonstrated a discrepancy between the finding of a 46,XY karyotype and the presence of female external genitalia with mild clitoromegaly, a small degree of posterior labial fusion, and the absence of uterus, fallopian tubes, and ovaries. The presence of mild clitoromegaly and posterior labial fusion allowed us to classify the syndrome as PAIS.
An impaired male phenotype development can be due to abnormalities in the synthesis of androgens or AR. Gene mutations and deletions of the AR can cause quantitative or qualitative protein defects that induce androgen resistance. In the patient studied, RT-PCR amplification of AR mRNA from in vitro cultured genital skin fibroblasts showed an amplification product whose length is compatible with lack of splicing of intron 6 and with a correct splicing of the intron immediately upstream of exon 6. The presence of a small amount of full-length AR suggests that the primary transcript can be partially spliced correctly even in the presence of a guanine to thymine substitution. Western blot analysis of genital skin fibroblasts extracts showed a receptor band of approximately 122 kDa, lower than the normal receptor (according to the manufacturer, the expected AR protein molecular mass is 132 kDa). By computer analysis of the intron sequence (15), we found a stop codon 79 bases after the splice site. In the absence of splicing, this would result in expression of a truncated receptor containing the amino acid sequence encoded by the first 6 exons plus 79 nucleotides and lacking amino acids encoded by exons 7 and 8, i.e. part of the C-terminal ligand-binding domain. In agreement with the RT-PCR results, Western blot analysis showed a faint band of the correct molecular mass. A 3-fold reduction in Bmax with a normal Kd in the binding studies also suggested that a reduced amount of the wild-type protein was present, with respect to that in a normal male control, which could explain the PAIS phenotype of the patient. The presence of both mutant and wild-type transcripts has been reported in PAIS patients by other researchers (21, 22, 23), and in two cases (21, 23) they concluded that the PAIS phenotype was produced from an insufficient amount of normal protein.
Splicing of precursor RNA involves cleavage of introns at their 5'-donor and 3'-acceptor splice sites followed by ligation of adjacent exons (24). Splicing takes place in a large complex, known as a spliceosome, composed of small nuclear ribonucleoprotein and multiple proteins (25). In the consensus sequence (26) of the donor splice site, guanine residue in position +5 is present in 85% of the sequences analyzed until now, and it is probably involved in the spliceosome assembly and the accuracy of cleavage (27, 28, 29, 30).
Point mutations altering mRNA splicing represent about 15% of all gene
mutations causing human genetic diseases (31), and splice donor sites
are more often involved than splice acceptor sites (32). Mutations in
position +5 of the splice donor site have been described to cause human
genetic diseases. Among these, five are transversions GT, as in our
study, and they result in the skipping of relative exons (33, 34, 35, 36) or
activation of a cryptic site (37). Alterations affecting AR gene
splicing are very rare (3%) (38), and in a few cases abnormal
splicing has been associated with AIS. Some of these cases concern
insertions, intronic deletions, or mutations outside the splicing
region (21, 22, 39, 40). In other cases, point mutations in the
consensus sequence donor splice site have been described, causing
excision of the exons 2, 3, 6, and 7 (39, 41, 42, 43) or activation of a
cryptic splice donor site in exon 4 (44). In contrast with previous
reports, our study shows neither exon skipping nor cryptic splice site
activation, but a complete retention of the mutant intron 6 containing
a stop codon in its sequence, which causes the production of a
truncated protein lacking the C-terminal portion of the AR encoded by
exons 7 and 8, that is presumably functionally inactive.
In AIS patients described to date, no clear-cut relationship is evident between clinical phenotype and the receptor abnormality. Indeed, the same mutation of the AR gene in the same family can result in clinical characteristics of CAIS or PAIS (45). Mutations producing stop codons or affecting the splicing region have been associated until now with a CAIS phenotype (20, 39, 41, 42, 43, 44). However, the patient studied here, albeit carrying a mutation in a splicing region that results in a stop codon, shows Quigley grade 5 PAIS.
Trifiro et al. (39) described a mutation very similar to that reported here, an A T mutation in +3 donor splice site of intron 6, that resulted in a CAIS phenotype. However, even though they could detect a small amount of normal mRNA, unlike in our report they were not able to detect AR protein expression.
In conclusion, the results of the present study shed more light on the impact of single point mutations in the AR gene. Our studies show that point mutations in the splicing region do not necessarily show complete penetrance and can result in a PAIS, rather then a CAIS, phenotype.
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Acknowledgments |
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Footnotes |
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Received October 12, 1999.
Revised May 18, 2000.
Accepted May 18, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylase gene of Japanese patients with 17-hydroxylase
deficiency. Hum Genet. 102:635–639.[CrossRef][Medline]
-hydroxylase/17,20-lyase deficiency. J Clin Endocrinol Metab. 83:199–202.1(I) collagen alters the efficiency of
pre-mRNA splicing and is associated with osteogenesis imperfecta type
II. J Biol Chem. 265:2262–2268.This article has been cited by other articles:
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