A Unique Exonic Splicing Mutation in the Human

0021-972X/01/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2001 by The Endocrine Society
Vol. 86, No. 6
Printed in U.S.A.
A Unique Exonic Splicing Mutation in the Human
Androgen Receptor Gene Indicates a Physiologic
Relevance of Regular Androgen Receptor
Transcript Variants*
´ -CARLOS HELLWINKEL, PAUL-MARTIN HOLTERHUS, DAGMAR STRUVE,
OLAF JOSE
CHRISTINE MARSCHKE, NICOLE HOMBURG, AND OLAF HIORT
Department of Pediatrics, Medical University of Lu¨beck, Lu¨beck, Germany D-23538
ABSTRACT
In a patient with partial androgen insensitivity syndrome (AIS), we
identified a single inherited presumably silent nucleotide variation
(AGC -⬎ AGT) in exon 8 (codon 888) of the AR gene. However, in the
patient’s genital skin fibroblasts, a considerably shortened transcript
of 5.5 kb (normal: 10.5 kb) was detected, which misses a part of exon
8 and a prominent portion of the 3⬘-untranslated region. The translation product includes eight missense amino acids from codon 886
onward followed by a premature stop codon. As shown by in vitro
expression analysis, the mutant protein lacks any residual function.
However, reverse transcribed PCRs and sequence data indicate the
existence of two additional splicing variants of 6.4 kb and 7.8-kb
length both in patient and normal control genital skin fibroblasts.
N
ORMAL SEXUAL DIFFERENTIATION in males is induced by androgen action. Signaling of androgenic
steroids in their target cells is enabled by the androgen receptor (AR), a phosphoprotein that complexes these ligands,
homodimerizes, and finally acts as a transcription factor on
genes, which induce virilization (1–3). The AR is encoded by
an X-chromosomal gene divided into eight exons (4). The
amino acid sequence encoded by exon 1 is partly involved in
transactivation of androgen target genes, the region encoded
by exon 2 and 3 enables DNA-binding, and the region encoded by exon 4 to 8 is involved in ligand binding (5).
Androgen insensitivity syndrome (AIS) is a common cause
for virilization disorders in patients with 46, XY karyotype.
The clinical spectrum of this disorder reaches from infertility
in males to a completely female phenotype (6). AIS is based
on the inability of androgen-dependent target tissues to react
to androgens. Most often, this is due to mutations within the
AR-gene (7–11). In most cases one defined point mutation
causing a single amino acid exchange or a premature termination induces defective DNA- or ligand-binding of the
AR (11).
Previously, in LNCaP cells derived from prostatic tissue
Received September 13, 2000. Revision received January 19, 2001.
Accepted February 28, 2001.
Address all correspondence and requests for reprints to: Olaf Hiort,
M.D., Department of Pediatrics, Medical University of Lu¨beck, Ratzeburger Allee 160, D-23538 Lu¨beck, Germany. E-mail: [email protected].
mu-luebeck.de.
*This work was supported by the Deutsche Forschungsgemeinschaft
(Hi 497/3–2,3 and Hi 497/4 –2 to O.H.)
These splicing variants comprise the complete coding region but a
shortened 3⬘-untranslated region. Thus, a distinct alternative premessegner RNA-processing event leading to two additional transcripts occurs generally in genital skin fibroblasts. In addition, this
process partially prevents aberrant splicing in the patient and produces a small fraction of normal, functionally intact AR-protein that
could explain the partial masculinization in this patient.
This first report of an exonic splicing mutation in the AR-gene
indicates a physiologic relevance of the regular AR-messenger RNA
variants with shortened 3⬘-untranslated regions and their functional
translation products in human genital development. (J Clin Endocrinol Metab 86: 2569 –2575, 2001)
and in prostatic tissue itself, the existence of a shortened
AR-transcript variant in addition to the full-length ARmessenger RNA (mRNA) has been reported (4, 12, 13). This
seems to be the result of a variable splicing event within the
3⬘-untranslated region (3⬘-UTR). To this date, it is unknown
whether transcript variants exist in other androgen target
tissues and are translated to functional AR-proteins in physiologically relevant amounts to influence the male
phenotype.
We present a patient with ambiguous genitalia bearing a
novel germline point mutation in exon 8 of the AR-gene. In
this context, we demonstrate the existence of two AR-transcript variants found in normal control and patient genital
skin fibroblasts (GSF) and discuss their physiological activity
and possible influence on the phenotype of our patient.
Materials and Methods
Patient
The propositus with a 46,XY karyotype and partial AIS [type 2b
according to the classification of Sinnecker et al. (6), corresponding to
type 3 according to Quigley et al. (3)] was studied at the age of 4 months.
The individual suffers from perineal hypospadias (grade IV), scrotum
bipartitum, undescended testes, and micropenis. Phallic enlargement
and rugation of the labia maiora provide clear clinical signs for residual
androgen action. Mullerian structures were not seen on ultrasound and
genitography. The family history was uninformative. At this time, baseline values for luteinizing hormone (0.7 U/L) and testosterone (1.0
nmol/L) were prepubertally low. After stimulation with hCG (1000 IU
daily for 5 days, im), serum-testosterone concentration rose markedly to
a value of 36.4 nmol/L (normal ⬎ 10 nmol/L), excluding a testosterone
biosynthesis defect and suggesting androgen insensitivity (14). A genital
skin biopsy was performed; at this time genital skin tissue was preserved
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2570
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HELLWINKEL ET AL.
for cell culture. Informed consent for detailed studies was given by the
mother.
Mutation analysis of the AR-gene
Genomic DNA from the patient and the mother was isolated from
peripheral blood leukocytes by standard procedures. The whole coding
region of the AR gene including all exon/intron boundaries was amplified by PCR in 14 segments using primers derived from published
sequences (15); then each fragment was tested for sequence variations
by single-strand conformation analysis (SSCA) as previously described
(7, 16). Briefly, PCR products were heat denatured, electrophoresed on
nondenaturating polyacrylamide (PAA) gels, and silver stained. Single
strands from PCR products that showed aberrant migration, compared
with normal controls, were cycle sequenced using the thermosequenase
fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP
(Amersham Pharmacia Biotech, Freiburg, Germany) and analyzed by an
ALF Express automated sequencer (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
Cell culture
Biopsy specimens from the patient’s genital skin were dissected mechanically and incubated in medium (DMEM-F12, 5% CO2, 10% charcoal-stripped FCS, antibiotics) at 37 C to grow fibroblasts. GSF obtained
from the foreskin of a normal prepubertal boy served as a control. Only
cells of the second and the third passage were used for AR-expression
studies.
RNA analysis, RT-PCRs, and Northern blots
Whole RNA from cultured GSF from the patient and the normal
control was isolated using Rneasy columns as indicated by the manufacturer (QIAGEN, Hilden, Germany). RNA was first quantified photometrically (absorbency at 260 nm measured in a DNA/RNA calculator
from Amersham Pharmacia Biotech). Then 5-␮L samples were electrophoresed and stained with ethidiumbromide on formaldehyde-denaturing 1% agarose gels to determine quality and integrity of RNA and
to test quantification results. If gels were planned to be blotted and used
for hybridization experiments (Northern blots; see below), 4-␮g RNA
per lane were loaded.
Extra long (XL) RT-PCRs for amplicons of 2 kb to 7 kb length and
RT-PCRs for amplicons smaller than 1 kb were performed on 1-␮g
whole-RNA samples. In both cases, RT was made by specific antisense
priming (primers see Table 1) with Superscript II reverse transcriptase
(Life Technologies, Inc., Eggenstein, Germany) following the manufacturer’s protocols.
For characterization of the AR coding region, fragments smaller than
1 kb were amplified by RT-PCRs with Ampli-TAq DNA-polymerase
(Perkin-Elmer Corp., Weiterstadt, Germany) using the primers indicated
in Fig. 1 and Table 1. Cycling conditions and PCR-solution compositions
were as described (10, 17). RT-PCR products were electrophoresed on 2%
agarose gels, stained with ethidiumbromide, and evaluated on a stan-
dard UV plate. Semiquantification of AR transcription was achieved by
competitive RT-PCRs as previously published (10, 17). Briefly, whole
RNA from patient and control GSF was first standardized for ubiquitous
ribosomal protein L7 transcription (semiquantitative RT-PCR with modified primers) (18). Then RNA samples were mixed with serially diluted
concentrations of artificial RNA-standard (shortened target) and submitted to RT-PCRs using the primers hARE1s and hARE4a (Table 1).
After electrophoresis on nondenaturating PAA gels and silver staining,
optical densities of PCR product signals (target: 479 bp; standard: 324 bp)
were evaluated by computerized densitometry (ImageMaster, Pharmacia). Signal optical densities of target (t) and standard (s) in each sample
were compared, resulting in dimensionless numbers used as relative
concentration equivalents of the transcript: A(t/s).
To investigate long complementary DNA (cDNA) fragments enclosing the exon 8/3⬘UTR link, we employed XL RT-PCRs. Amplicons were
between 2 kb and 7 kb long; for primer sequences and locations, see Fig.
1 and Table 1. For this purpose, the GeneAmp XL PCR kit (Perkin-Elmer
Corp.) was used according to the producer’s recommendations. Initial
examination of XL RT-PCR products was made after electrophoresis on
1% agarose gels and ethidiumbromide staining on a UV plate. Before
sequencing, products were isolated from the gel and finally purified by
QuiaQuick columns (QIAGEN) twice.
Northern blots and probe generation were performed as previously
published (10, 17). In brief, digoxigenin-labeled AR-RNA probes were
generated by RT-PCR with an antisense primer tailed by a T7-RNApolymerase recognition sequence. After purification, the product was in
vitro transcribed by T7-RNA-polymerase (Promega Corp., Heidelberg,
Germany) using digoxigenin-labeled rNTPs (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. Finally DEPC-water was added to a final volume of 100 ␮L.
Northern blot filters were prehybridized for 1–2 h and then hybridized
with 10 mL Easy-hyb solution (Roche Molecular Biochemicals) and the
probe (3 ␮L) at 68 C overnight. Stringent washings were performed at
68 C with 0.1 ⫻ SSC/0.1% SDS for 2 ⫻ 15 min. Chemoluminescence
signals of hybridized probes were developed following the Digoxigenin
users guide for Northern blots (Roche Molecular Biochemicals).
Western immunoblots
Western blots for AR proteins expressed in the patient and the normal
control GSF strains were performed as described previously (19, 20).
Specific anti-AR antibodies were a generous gift of Dr. A. O. Brinkmann
(Erasmus University, Rotterdam, The Netherlands). In brief, GSF were
first lysed to isolate AR proteins from whole-cell lysates by immunoprecipitation using the monoclonal anti-AR antibody F39. Subsequently,
the samples were size fractionated by SDS-PAGE and blotted on cellulose-nitrate membranes. These were first incubated with the primary
antibody SP061 directed against the amino acids 301–320 of the AR (19).
Then a second incubation with an antirabbit-peroxidase conjugate was
performed. Detection of AR proteins was accomplished by
chemiluminescence.
TABLE 1. Primers employed for RT PCR amplification
Primer
HARE1s
HARE6s
HARE7s
HARE4a
hARE8a
hAR3⬘UTRa
Sequence and 5⬘ 3 3⬘ annealing position (Ref.)
5⬘-TGG ATG GAT AGC TAC TCC GG-3⬘
nucleotides 1654 –1673 (15)
5⬘-TAC CGC ATG CAC AAG TCC CGG-3⬘
nucleotides 3348 –3368 (15)
5⬘-CTC ACC AAG CTC CTG GAC TC-3⬘
nucleotides 3917–3937 (15)
5⬘-ACT ACA CCT GGC TCA ATG GC-3⬘
nucleotides 2845–2826 (15)
5⬘-GAG GAG TAG TGC AGA GTT ATA A-3⬘
nucleotides 4287– 4266 (15)
5⬘-CAG AAC ACT AGC GCT TGG AG-3⬘
nucleotide 10333–10313 (13)
Used fora
Competitive RT PCRs
RT PCRs
XL RT PCRs
XL RT PCRs
Competitive RT PCRs
Specific RT-priming
RT PCRs
Specific RT-priming
XL RT PCRs
Specific XL RT-priming
Information in this column can also be found in text and in Fig. 2.
a
Corresponding nucleotide positions are indicated as in the published AR-gene- or -transcript sequences. Note that the primer hAR3⬘UTRa
binds 43 bases upstream of the first polyadenylation site of the AR transcription unit.
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ANDROGEN RECEPTOR TRANSCRIPT VARIANTS
2571
FIG. 1. Complete AR transcription unit
(10.5 kb) with enlarged open reading
frame (amino acid [aa]-coding region),
primer annealing areas, and expected
(XL) RT-PCR product lengths. Primer
sequences are given in Table 1.
Androgen-binding studies
Androgen-binding assays were performed according to previous protocols (20, 21). In brief, confluent GSF cultures from either the patient or
the control subject were incubated with media containing various concentrations of (3H)R1881 (17␤-hydroxy-17␣-(3H)-methyl-4,6,11-oestrotrien-3-one (NEN Life Science Products, Boston, MA) in either the presence or absence of a 200-fold molar excess of unlabeled ligand. After 1
hour incubation at 37 C, aliquots of 50 ␮L culture medium were taken
for measurements of total counts. Cells were washed, lysed, and submitted to liquid scintillation counting; 100-␮L samples were reserved for
protein determination. Results were evaluated by computerized Scatchard analysis.
AR expression plasmids
The sequence of the shortened AR-transcript specific for the patient’s
GSF was introduced into the human AR expression vector pSVAR0 (21).
First, RT-PCRs were applied on whole RNA from the patient GSF with
the sense-primer hARE 1s (Table 1 and Fig.1) and the antisense-primer
hAR-BFa (5⬘-CCT CTT AAG GGA TCC AGG TCA CAA GTA CAT GGC
ATC -3⬘). This primer is unique for a part of the aberrant sequence in the
patient-specific AR-mRNA (in italics) and encloses the premature stop
codon (underlined); the primer is tailed at the 5⬘-end by a sequence
containing a BamHI recognition site (bold typed) for later cleavage. The
PCR mixture contained 1 ␮L cDNA (first strand prepared for XL RTPCRs), 200 ␮m dNTPs, 1⫻ PCR-buffer (pH 9.0), 1.0 mm MgCl2, 20pmole
each sense and antisense primer and 1U Ampli-TAq DNA-polymerase
(Perkin-Elmer Corp.). Cycling conditions were: 75-sec denaturation at 94
C, 90-sec primer annealing at 52 C, and 120-sec primer extension at 72
C for 35 cycles. After testing the success of the experiment on a 2%
agarose gel, the RT-PCR product was immediately cloned into the
PCR2.1 vector (TA cloning kit, Invitrogen, Leek, The Netherlands). After
verifying the correct sequence by plasmid sequencing (Seqlab, Go¨ttingen, Germany), PCR2.1 (comprising the patient-specific RT-PCR product as insert) and pSVAR0 were digested with BamHI and Asp1 (Roche
Molecular Biochemicals) and subsequently purified from agarose gels.
Ligation of the BamHI-Asp1 insert into the pSVAR0 vector was performed using T4-DNA-ligase according to the manufacturer’s instructions (Invitrogen). The ligation product was transformed in DH5␣-Escherichia coli bacteria and single clones were purified by the QIAprep spin
miniprep kit (QIAGEN). The correct plasmid sequence (further called
pSVAR3⬘Del) was verified again.
Transient transfections
Either pSVAR0 or pSVAR3⬘Del were transiently transfected in eukaryotic chinese hamster ovary cells using the calcium phosphate precipitation method (23) with only minor changes as described previously
(20). Activation of cotransfected androgen inducible MMTV-luciferase
reporter gene was determined either in the absence of hormone or in the
presence of 0.1-, 1.0-, and 10.0 nm dihydrotestosterone, respectively. All
transfections were performed in triplicate, and three independent experiments were carried out. Transfection efficiency was determined by
cotransfection of the constitutively expressed pRLSV40 Renilla luciferase plasmid (Promega Corp.). Firefly and Renilla luciferase activity
were measured using the dual luciferase reporter gene assay (Promega
Corp.).
Results
Analysis of the AR-gene
SSCA of PCR products amplifying exon 8 displayed an
aberrant migration pattern on the PAA gel, compared with
normal controls. Sequencing revealed a transition of cytosine
to thymidine (AGC 3 AGT) in the third position of codon
888. The mutation has no influence on the predicted amino
acid coding sequence itself as both triplets code for serine.
The mother of the patient was found to be heterozygous
carrier of this variation. By SSCA of the whole coding region
and additional sequencing of exons 4 to 8 of the AR-gene, we
could not detect any additional mutation. The polymorphic
trinucleotid repeats within the first exon were determined to
carry 21 CAG-repeats and 24 GGN-repeats.
Studies of the AR-transcript
By the Northern blot shown in Fig. 2A, we could prove the
existence of an AR-transcript of approximately 5.5 kb length
in the patient’s whole RNA. This is considerably shorter than
the wild-type AR-transcript found in whole RNA from control GSF (10.5 kb). An additional band at 4.9 kb in patient and
control RNA represents a commonly observed AR-mRNA
degradation product (13). UV luminescence and ratio of
ethidium-bromide stained 28S- and 18S-rRNA bands on the
gel and L7 housekeeping gene transcription in both patient
and control material were similar indicating equivalent RNA
quantity and integrity. Then competitive RT-PCRs of an exon
1– 4 amplicon of the AR-transcript (primers hARE1s and
hARE4a; see Fig. 1 and Table 1) with 0.02-, 0.1-, and 0.5attomole competitor/␮g RNA were applied. As shown in
Fig. 2B, calculated competitor amounts needed to obtain an
A(t/s) value of 1.0 (indicating similar amounts of target and
standard amplificates) were 0.06 and 0.18 attomole per ␮g
RNA from the patient and the normal control, respectively.
Comparison of these values revealed that the AR-transcript
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2572
HELLWINKEL ET AL.
JCE & M • 2001
Vol. 86 • No. 6
FIG. 2. Effects of the patient’s mutation on the AR-transcript. A, Northern blots on each 4-␮g whole RNA from patient and normal control GSF
hybridized to an AR-RNA probe. Bold-typed kilobase pair values indicate AR-transcript lengths. Note that the patient’s AR-transcript is shorter
than wild-type AR-mRNA. Signals at 4.9 kb most likely denote AR-mRNA degradation products. B, Relative transcript amounts in patient and
normal control GSF determined by competitive RT-PCRs. A(t/s) values per microgram whole RNA from patient (black curve) and control (gray
curve) are depicted logarithmically as a function of the applied competitor amount (see also text). Boxed, italic values under the graph represent
interpolated competitor concentrations needed to achieve balanced target-to-standard optical densities (A[t/s] ⫽ 1); they give information about
relative transcript amounts. C, RT-PCR products on RNA from patient and control GSF obtained with primers hARE1s and hARE8a (spanning
all exon boundaries). The patient and the normal control amplificates are of equal length. Top bands of the molecular weight marker (␾ 174;
HaeIII -digest) are 1353 and 1078 bp long. D, XL RT-PCR products on RNA from patient and control GSF obtained with primers hARE7s and
3⬘UTRa (comprising the major part of the 3⬘UTR). Boldface kb values represent specific products. Note that the patient’s material lacks the
full-length amplificate but shares two shortened amplificates with the control and displays one additional band at 1.7 kb. The band sizes of
the molecular weight marker (1-kb ladder) are: 1.64, 2.04, 3.05, 4.07, 5.09, 6.11, 7.13 [. . . ] kb.
level in patient GSF amounts to approximately 30% of that
in GSF from the equally aged normal control. RT-PCRs of an
exon 1– 8 amplicon of the AR-mRNA (primers hARE1s and
hARE8a) including all exon boundaries and the mutation
region (Fig. 1) yielded products of equal length in samples
with normal and patient whole RNA as shown in Fig. 2C. By
these RT-PCRs and experiments with other mutation-flanking primer pairs binding sequences within the coding region,
no additional products could be demonstrated in patient
material (not shown). XL RT-PCRs with primers flanking the
mutation site and the major part of the 3⬘-UTR (primers
hARE6s and hAR3⬘UTRa; Fig. 1) lead to the generation of
three products (3.75 kb, 2.55 kb, and 1.95 kb) that are shorter
than the wild-type amplicon (6.42 kb). To determine whether
these products were specific, XL PCRs with primers hARE7s
(hybridizing 264 bp downstream of hARE6s; Fig. 1) and
hAR3⬘UTRa were performed on 10,000-fold diluted samples
from gel-purified initial XL RT-PCR products. As demonstrated in Fig. 2D, the three products from the first XL RTPCR must be derived from the AR-transcript because they
are approximately 260 bp shorter (3.5 kb, 2.3 kb, and 1.7 kb).
In the patient whole RNA, a full-length wild-type XL RT-PCR
product (6.42/6.16 kb) could not be demonstrated. In contrast, on normal control RNA, a faint band specific for the
full-length amplicon could be detected after a seminested XL
RT-PCR with primers hARE7s and hAR3⬘UTRa. However,
the specific 3.5-kb and 2.3-kb seminested XL PCR products
found in patient material were also generated on normal
control whole RNA (Fig. 2D). In contrast, the 1.7-kb amplificate could not be reproduced on normal material. Additional
smears and faint bands (visible on the gel in Fig. 2D) were
not mirrored by corresponding signals on electrophorated
samples of the first XL RT-PCR, indicating those signals to be
unspecific.
Finally, exon 8 sequences of the XL RT-PCR product derived from the patient-specific AR-transcript (1.96 kb/1.7 kb)
and one shortened amplificate found in patient and normal
material (2.55 kb/2.3 kb) were sequenced. As illustrated in
Fig. 3, the product derived from the 1.7-kb patient-specific
AR-transcript displays a deletion beginning 5 bp upstream of
the mutant T in codon 888 shown below in the 2.3-kb short
regular AR-transcript (Fig. 3). The last sense codon 886 (Met)
is merged to a downstream sequence derived from the 3⬘UTR
coding for 8 missense amino acids and followed by a premature stop-codon. Upstream of codon 886, sequencing up
to exon 4 on the subcloned patient-specific amplificate revealed correct amino acid coding. In contrast, the 2.55 kb/2.3
kb amplificate exhibits the complete and correct coding sequence of exon 8 of the AR-gene.
To examine whether RNA from normal GSF contains small
amounts of patient-specific AR-transcript, we designed a
composite-primer (hAR-BFa) specific for a fraction of the
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ANDROGEN RECEPTOR TRANSCRIPT VARIANTS
2573
FIG. 3. Nucleotide sequences (from
codon 884 in exon 8 going downstream)
and resulting amino acid coding from
the patient’s 1.7- and 2.3-kb XL RTPCR products from Fig.2D. The shared
wild-type sequence appears bold typed;
missense nucleotides and amino acids
are in italics. Stop-codons are underlined. Note that the 2.3-kb XL RT-PCR
product displays correct wild-type-AR
amino acid coding, and in the 1.7-kb
product a missense amino acid sequence is encoded and a premature termination codon is introduced at codon
position 895.
FIG. 4. Western blot from patient and normal control GSF detected
by labeled anti-AR antibodies. Kilodalton values indicate sizes of
normal AR-protein isoforms (for further information see text).
Patient-specific AR-proteins appear to be slightly shorter.
3⬘UTR sequence merged to codon 886 in the patient’s aberrant AR-mRNA shown in Fig. 3. Using this primer and the
primer hARE1s, we could reproducibly amplify a specific
(1.13 kb) RT-PCR product on patient material but not on
normal control RNA. This demonstrates the absence of
patient-specific AR-transcripts in normal GSF.
Studies of AR translation
In the patient’s GSF, we found an AR-protein that was
slightly smaller than the normal AR 110/112 kDa doublet
(Fig. 4). Correspondingly, a lighter AR-protein (87 kDa) that
most probably is the result of a downstream translationinitiation at Met189 (4) found in normal material also appeared to be smaller in the patient sample.
Scatchard analyses of androgen-binding data on the patient’s GSF revealed a normal Kd of 0.093 nm and a Bmax of
4.8 fmole/mg protein, which indicate a very low level of
otherwise normal androgen binding. Expectedly, the control
cell line showed normal Kd- and Bmax-values (0.073 nm and
46.11 fmole/mg protein, respectively).
In transient transfection assays, the pSVAR0 plasmid representing the wild-type androgen receptor showed a concentration dependent activation of the androgen inducible
MMTV-luciferase reporter gene (Fig. 5). However, the plasmid construct pSVAR3⬘Del reflecting the deleted patientspecific translation product failed to induce the reporter
gene, even in the presence of supraphysiological concentra-
FIG. 5. Androgen-induced transactivation capacity of the patientspecific AR-protein (cDNA cloned as “pSVAR3⬘Del”) in comparison
with a normal control (wild-type-AR cDNA clone “pSVAR0”). The
induction of luciferase activity calculated in relation to basal activity
found in the absence of ligand of three independent experiments is
depicted as a function of dihydrotestosterone incubation.
tions of dihydrotestosterone. This demonstrates the absence
of any transactivation activity of the patient-specific ARtranslation product.
Discussion
So far, only few mutations that lead to extensive structural
aberrations of the AR-transcript or -protein have been described (11). In these cases, exonic mutations normally lead
to the introduction of a premature stop-codon, and intronic
alterations within or nearby splice sites cause aberrations of
the splicing process. First, exons adjacent to the mutated
region can be completely absent in mature mRNA (10, 24, 25).
Second, mutations can lead to the generation of various transcripts with either unaltered normal sequence, inserted nucleotides or absent exons (26, 27). This phenomenon can be
explained by the activation of cryptic splice sites.
The mutation presented here leads to some extent to the
generation of a shortened transcript in the patient (Fig. 2).
This patient-specific AR-transcript of approximately 5.5 kb
length indicates alternative splicing, caused by the activation
of a cryptic splice-donor site within exon 8 in which the
wild-type-sequence “. . . GGUGAGC. . . ” is changed by the
mutation to “. . . GGUGAGU. . . ”. The mutated sequence is
similar to the donor splice-site consensus sequence “. . . exon
GGUAAGU intron. . . ” In aberrant AR-pre-mRNA splicing,
the binucleotide “GU” now serves as splicing-signal leading
to false intron-demarcation of the last part of exon 8 and a
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2574
HELLWINKEL ET AL.
prominent fraction of the 3⬘UTR. In connection with an assumed acceptor splice-site located within the 3⬘UTR, the
splicing process merges a downstream 3⬘UTR-fragment to
codon 886, removing the internalized sequence. By this
event, a sequence coding eight missense amino acids and a
premature stop codon follows codon 886 (Fig. 3).
Alternative AR-mRNA splicing has been reported to occur
in the prostatic cancer cell line LNCaP and in cells from
prostatic tissue (4, 12, 13). In both cases, only 3⬘UTR sequences seem to be altered. Faber et al. (13) described a
sequence surrounding position 8685 in the 3⬘UTR: “. . . intron
CTTTAAC[N (27)]CAGATCA exon. . . .” It conforms to the
acceptor splice-site consensus sequence and most probably
serves as an alternative AR pre-mRNA splice site in LNCaP
cells. In our patient, the aberrant splicing event leading to the
formation of the patient-specific AR-transcript employs the
new donor splice site behind codon 886 (located 3759 bp
downstream of the transcript’s 5⬘-end) and presumably the
above-mentioned sequence at position 8685 as acceptor
splice site. As depicted in Fig. 6, this will lead to an ARmRNA of approximately 5.57 kb length (deleted of 4.93 kb),
matching with the 5.5-kb patient-specific AR-transcript
found in the Northern blot. This consideration is further
supported by the finding that the first four nucleotides of the
3⬘UTR sequence spliced to codon 886 (ATG) in the patientspecific AR-mRNA are identical to the first “exonic” bases
following the putative acceptor splice site given above.
In patient material, we could not find indications for the
existence of a full-length AR-transcript by RT-PCRs or
Northern blotting (Fig. 2, A and D). Interestingly, seminested
XL RT-PCR products of 3.5 kb and 2.3 kb length were gen-
JCE & M • 2001
Vol. 86 • No. 6
erated on whole RNA from patient and normal control GSF
(Fig. 2D). The 2.3-kb amplificate includes the complete 3⬘-end
of the AR-mRNA coding sequence (Fig. 3) with a deletion
exclusively restricted to the 3⬘UTR. Subtraction of this
3.86-kb deletion from the full-length transcript (10.5 kb) unveils a transcript size of approximately 6.4 kb. Correspondingly, the second shortened AR-transcript missing approx.
2.66 kb is about 7.8 kb long and most likely also comprises
the complete coding region. As illustrated in Fig. 6, we believe that both shortened transcripts are the result of regular
but rare splicing events applying the acceptor splice-site at
position 8685 and 2 donor splice sites lying upstream but still
within the 3⬘UTR. The 7.8-kb transcript seems to be identical
to the similarly long 3⬘UTR-shortened AR-mRNA reported
by Faber et al. (13) for LNCaP cells or the alternative ARtranscript described by Lubahn et al. (4) for prostatic tissue.
In our experiments, both shortened wild-type AR-transcripts
are not visible by Northern blotting indicating very low
concentrations in control GSF, compared with the full-length
transcript. This may explain the lack of other reports for
alternative AR splice products in GSF or other androgen
target tissues. Possibly, the 7.8-kb and/or 6.4-kb shortened
wild-type AR-transcripts may exist in many or even all
androgen-sensitive tissues.
The existence of shortened wild-type AR-transcripts in
patient material may be explained by the following consideration (see also Fig. 6): During the splicing process, patientspecific AR-mRNA formation may be favored because of the
activated strong splice-donor site in exon 8. However, a small
portion of AR-pre-mRNA is spliced applying both donorsplice sites and the wild-type acceptor splice site described
FIG. 6. Illustration of hypothesized normal and aberrant AR-transcripts and the derived XL RT-PCR products. Also, putative splicing sites and
effects are indicated. Transcripts are represented by full-size, XL RT-PCR products by thinner rows. Splice sites within the 3⬘UTR of the
full-length AR-mRNA are depicted as small gray marks. The site of the mutation in codon 888 of the patient’s AR-gene is marked by an “x”
within the full-length AR-mRNA. At the left side, the origin of GSF, in which the respective transcripts can be found, is designated. ORF, Open
reading frame; ⌬, deletion due to the respective splicing effect.
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ANDROGEN RECEPTOR TRANSCRIPT VARIANTS
above within the 3⬘UTR leading to the generation of shortened wild-type AR-transcripts. Thus, as the acceptor splice
site is eliminated, subsequent aberrant splicing cannot occur.
This hypothesis matches the results on the translation
level. The Western blot (Fig. 4) demonstrates that the patientspecific AR-transcript is translated, resulting in a slightly
shortened protein, compared with the wild-type AR (110/
112 kDa or 87 kDa) found in control GSF. However, normal
AR-protein was not discriminated in patient material by this
approach; this could originate in only small quantities of
normal AR displaying a hardly or undetectable band, which
additionally may be covered by the nearby laying band of
aberrant AR-protein. In contrast, androgen-binding analysis
in patient GSF indicated the existence of a very small fraction
of protein with normal androgen-binding properties. Therefore, we assume that the normal AR-protein fraction in patient GSF is too small to be detected by our Western blot, and
it is above the detection level in the androgen-binding assay.
Function of the patient-specific mutant AR-protein was excluded by androgen-induced transactivation in cotransfection assays (Fig. 5). This was expected because in other studies completely absent androgen-binding capacity is observed
for AR-proteins bearing a premature termination in the
hormone-binding region (3).
Therefore, we conclude that the clinically and biochemically apparent residual androgen action in the presented
patient is most likely mediated by the translation products of
small quantities of functional shortened wild-type AR-transcripts. It remains to be investigated whether the normal,
alternative splicing effects described here may individually
affect the variable genotype-phenotype correlation observed
in AIS.
Acknowledgments
We are grateful to Dr. Hartmut Merz for his friendly permission to
use parts of the laboratory equipment from the Department of Pathology
of the Medical University of Lu¨beck and especially to Anke Mu¨ller for
her excellent technical counseling. We thank Timo Gaiser for his advice
allowing successful cloning of the patient-specific AR-cDNA. We further
thank Nicole Getschmann for her excellent technical assistance.
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