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Original Articles: Hormones and Reproductive Health |
Department of Pediatrics, Medical University of Lübeck, Lübeck, Germany D-23538
Address all correspondence and requests for reprints to: Olaf Hiort, M.D., Department of Pediatrics, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: hiort{at}paedia.ukl mu-luebeck.de.
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. These splicing variants comprise the complete coding region but a shortened 3'-untranslated region. Thus, a distinct alternative pre-messegner 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.
NORMAL 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, 2, 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, 8, 9, 10, 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 and in prostatic tissue itself, the existence of a shortened AR-transcript variant in addition to the full-length AR- messenger 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 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-µL 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-µg 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-µg
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.
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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 standard
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).
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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-RNA-polymerase 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 µL. 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 µL) at 68 C overnight. Stringent washings were performed at 68 C with 0.1 x SSC/0.1% SDS for 2 x 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.
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
(17ß-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 µL culture medium
were taken for measurements of total counts. Cells were washed, lysed,
and submitted to liquid scintillation counting; 100-µL 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 µL cDNA (first strand prepared
for XL RT-PCRs), 200 µM dNTPs, 1x 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, Göttingen, 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 
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 0.5-attomole competitor/µg 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 µg RNA from the patient and the normal
control, respectively. Comparison of these values revealed that the
AR-transcript 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 RT-PCR 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.
|
, 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.
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. 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
translation-initiation at Met189 (4) found in
normal material also appeared to be smaller in the patient sample.
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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 patient-specific translation product
failed to induce the reporter gene, even in the presence of
supraphysiological concentrations of dihydrotestosterone. This
demonstrates the absence of any transactivation activity of the
patient-specific AR-translation product.
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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
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 AR-mRNA 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
patient-specific AR-mRNA are identical to the first "exonic" bases
following the putative acceptor splice site given above.
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, A
and D). Interestingly, seminested XL RT-PCR products of 3.5 kb and 2.3
kb length were generated 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 AR-transcript
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, patient-specific 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
donor-splice sites and the wild-type acceptor splice site described
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 patient-specific
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 Lübeck and especially to Anke Müller 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.
Footnotes
1 This work was supported by the Deutsche Forschungsgemeinschaft (Hi
497/3–2,3 and Hi 497/4–2 to O.H.) 
Received September 13, 2000.
Revised January 19, 2001.
Accepted February 28, 2001.
References
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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.
, deletion due to the
respective splicing effect.