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J. Biol. Chem., Vol. 275, Issue 26, 19964-19969, June 30, 2000
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§¶,
,§, and
From the Department of Molecular Medicine,
§ Division of Neurology, and Division of Clinical
Neurophysiology, Karolinska Hospital, Stockholm 171 76, Sweden and
** Division of Neuroscience and Psychological Medicine and
Division of Surgery, Anaesthesia, and
Intensive Care, Imperial College School of Medicine, Charing Cross
Hospital, London W6 8RP, United Kingdom
Received for publication, February 24, 2000, and in revised form, March 27, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Myotonic dystrophy is caused by a
CTGn expansion in the 3'-untranslated region of a
serine/threonine protein kinase gene (DMPK), which is
flanked by two other genes, DMWD and SIX5. One
hypothesis to explain the wide-ranging effects of this expansion is
that, as the mutation expands, it alters the expression of one or more of these genes. The effects may vary in different tissues and developmental stages, but it has been difficult to develop these hypotheses as the normal postnatal developmental expression patterns of
these genes have not been adequately investigated. We have developed
accurate transcript quantification based on fluorescent real-time
reverse transcription-polymerase chain reaction (TaqMan) to develop
gene expression profiles during postnatal development in C57Bl/10 mice.
Our results show extensive independent postnatal regulation of the
myotonic dystrophy-locus genes in selected tissues and demonstrate
which are the most highly expressed of the genes in each tissue. All
three genes at the locus are expressed in the adult lens, questioning a
previous model of cataractogenesis mediated solely by effects on
Six5 expression. Additionally, using an in vivo
model, we have shown that Dmpk levels decrease during the
early stages of muscle regeneration. Our data provide a framework for
investigation of tissue-specific pathological mechanisms in this disorder.
Myotonic dystrophy
(DM1)1 is an autosomal
dominant neuromuscular disorder with very variable symptom
presentation. Anticipation (a decrease in the age of onset with
transmission) is often seen in DM1 and is usually accompanied by
increased symptom severity (1). The most severe manifestation of the
disease is the congenital form, in which newborns present with
generalized skeletal muscle hypotonia. In marked contrast, the most
minimally affected patients may simply develop late-onset cataracts
(clinical presentation reviewed in Ref. 1). The mutation underlying DM1
is a CTGn expansion on chromosome 19 (2-7). In the normal
population the repeat is between 5-37 copies, but in the DM1
population this may reach several kilobases in peripheral blood
leukocytes (8). The repeat is mitotically and meiotically unstable, and
there is a broad but not absolute correlation between the expansion size and symptom severity, providing a molecular basis for anticipation (9-11).
There is no consensus on how the mutation causes the complex and varied
clinical manifestation in DM1. A number of hypotheses have been
advanced, including the titration and sequestration of specific
RNA-binding proteins (12-14) or the formation of abnormal chromatin
structures (15-17). Most attention has focused on the effects of the
expansion on nearby genes. The CTG repeat lies in the 3'-untranslated
region of a serine/threonine protein kinase (DMPK) (3). A
homeodomain gene of the SIX family (SIX5,
formerly DMAHP) (18) lies less than 1 kilobase from the 3'
end of DMPK, and a WD-repeat gene (DMWD, formerly
59) (19) terminates less than 1 kilobase from the 5' end of
DMPK. It has been hypothesized that the expansion may
influence the expression of these DM1-locus genes and that this effect
could vary in different tissues and at different developmental stages.
Allele-specific decreases in the expression of the DM1 locus genes in
tissues derived from DM1 patients have been reported (20-23), although
a recent study reported that overall expression of the transcripts fell
within the control population range (24).
If gene dosage at the DM1-locus contributes to the development of the
phenotype, its effects may vary in different tissues, depending on the
basal expression of each gene. Existing data on expression of the
DM1-locus genes is mainly based on relatively insensitive techniques
e.g. Northern blotting, which is insufficient to detect
SIX5 expression in most tissues or non-quantitative RT-PCR.
Additionally, there have been no truly systematic studies addressing
the alterations in expression occurring postnatally, which may be
significant given the very different presentations in the congenital
and adult forms of the disorder and the late-onset of some of the symptoms.
To address this we have examined the expression of the DM1 locus genes
in a range of tissues from neonatal and mature mice to generate a
spatial and temporal map of expression from this highly conserved
locus. To obtain genuinely quantitative data for a large number of
samples, it is essential to use a technique that is sensitive,
accurate, and has high throughput. The TaqMan system (Perkin-Elmer),
which measures fluorescence in real time, fulfils all these
requirements and was used throughout.
Additionally, it has been reported that in tissue culture systems high
levels of DMPK or its 3'-untranslated region inhibit differentiation of myoblasts to mature myotubes (25, 26). It was
unclear how, if at all, this related to muscle physiology in
vivo. By using an in vivo experimental model of
skeletal muscle regeneration in mice, we have extended the
physiological significance of these findings.
Mouse Tissue Samples--
Wild type C57B1/10 mice were
sacrificed by cervical dislocation, and samples were removed,
snap-frozen in liquid nitrogen, and stored at In Vivo Regeneration Samples--
Skeletal muscle regeneration
was induced in the tibialis anterior muscle of 4-week-old male C57Bl/10
mice. 50 µl of 1.2% BaCl2 in Hanks' balanced salt
solution were injected into the right tibialis anterior; 50 µl of
Hanks' balanced salt solution alone were injected into the left
tibialis anterior as a control. The mice (3 per time point) were
sacrificed at 3, 10, and 25 days post-injection, and the tibialis
anterior muscles were removed. One-third of each muscle was snap-frozen
in liquid nitrogen for TaqMan, and the remaining two-thirds were
mounted in Bright Cryo-M-Bed (Bright Instrument Co., Ltd.) and frozen
in isopentane cooled to mRNA Extraction and cDNA Synthesis--
Cytoplasmic
poly(A) mRNA was isolated from 10-40 mg of tissue using the
Invitrogen Micro Fast Track mRNA isolation kit. 70% of the
isolated mRNA was used as a template for the production of
first-strand cDNA using random primers (cDNA Cycle Kit,
Invitrogen). The cDNA was resuspended in 50 µl of nuclease-free
sterile distilled water. 14 µl of each cDNA was mixed with 56 µl of sterile distilled water, and this diluted preparation was used
as the template for each TaqMan assay for the 4 genes. The remaining
30% of the mRNA was used as the template for a no-reverse
transcriptase control.
TaqMan Assays--
The TaqMan system is based on Taq
polymerase 5'-3' nuclease activity (27), which cleaves a dually labeled
non-extendible TaqMan probe designed to hybridize to a sequence between
the forward and the reverse primer for every particular amplicon. The
probe has a quencher dye on its 3' end and a reporter dye at the 5' end. Fluorescence emission from the reporter is quenched by the quencher dye until nuclease degradation during the extension phase of
the PCR separates the two dyes and enables detection of the reporter
dye fluorescence (28, 29). Primers and probes for the four different
amplicons were designed using the Primer Express (Perkin-Elmer) and
Oligo 5.0 software (National Biosciences). The amplicons were designed
to have one primer covering an exon-exon boundary. Primer sequences
(Interactiva, Ulm, Germany; high performance liquid
chomatography-purified): Dmpk (exons 2/3),
5'-cccattgcagcaaggcttaa-3' and 5'-tcaccaccgctacctcgc-3';
Dmwd (exons 2/3), 5'-tgccacagagaccatttccc-3' and
5'-tgtcaatcagccgctcttca-3'; Six5 (exons b/c),
5'-agtcctgtgaaggtggcagc-3' and 5'-gaggaagtttcctggagcagtg-3';
Tbp (exons 6/7), 5'-tgggcttcccagctaagttc-3' and
5'-ggaaataattctggctcatagctactg-3'. Probe sequences (Perkin-Elmer): Dmpk, 5'-cccacgcccgatcaccttcaa-3'; Dmwd,
5'-cctcatcaagaaagacaccagcaagctattc-3'; Six5,
5'-cagtcactcccacactagagtttatcaggtgca-3'; Tbp,
5'-tccccaccatgttctggatcttgaagtcta-3'. Melting temperatures of the
primers were 58-60 °C, and melting temperatures of the probes were
67-70 °C. Amplicon sizes: Dmpk, 100 base pairs;
Dmwd, 113 base pairs; Six5, 114 base pairs, and Tbp,136 base pairs. The same preparations of primers and
probes were used in all experiments. Reporter dyes and quencher dyes for all probes were 6-carboxyfluorescein (FAM) and
6-carboxytetramethylrhodamine (TAMRA), respectively. The TaqMan PCR
reaction conditions were: 2 min at 50 °C, 10 min at 95 °C, then
40 cycles each of 15 s at 95 °C and 1 min at 60 °C on
MicroAmp Optical 96-well plates, covered with MicroAmp Optical caps.
Each plate contained triplicates of the test cDNA templates, a
standard curve for the individual amplicon, and no-template controls
for each reaction mix. The precise composition of each reaction mix was
determined following initial optimization using cloned templates. All
samples contained 1× TaqMan Buffer A (Perkin-Elmer) and 5 mM MgCl2, 200 µM each dATP, dCTP,
and dGTP, 400 µM dUTP, 100 nM TaqMan probe,
0.625 units of AmpliTaq Gold polymerase, and 0.25 units of AmpErase
uracil-N-glycosylase in a 25-µl volume using 5 µl of
diluted cDNA. For Tbp, Dmwd, and Six5, both primers were used at 300 nM; for
Dmpk, the forward primer was used at 900 nM, and
the reverse primer was used at 50 nM.
During the assay, the linear increase in fluorescence signals from the
reporter dye was collected every 7 s by an Applied Biosystem Inc.
Prism 7700. Raw data was analyzed using Sequence Detector Software
1.6.3, and the
To obtain absolute quantification, as desired in this study, it is
essential to incorporate standard curves for every assay. Standard
curves for each amplicon were plotted from 5 different concentrations
of standards run in triplicate and were considered acceptable if the
correlation coefficient exceeded 0.98 (correlation coefficient and S.D.
among the triplicates calculated using Sequence Detector Software). For
Ct values <30, triplicates were not allowed to differ by more than 0.3 units. For Ct values >30 the variation was not allowed to exceed 0.5 Ct. For any samples exceeding these limits, the entire TaqMan assay was
repeated at least 3 times.
The expression data for all samples were normalized to the levels for
an established (30) housekeeping gene Tbp (TATA-binding protein). Transcript levels of this gene remain constant, with alterations in Tbp protein levels in certain adult tissues
mediated by translational/post-translational mechanisms (31). The only known exception to this is adult testis in which there are higher levels of Tbp mRNA (32), necessitating a conversion
factor of 18. The copy numbers for the DM1-locus genes were normalized
to the copy numbers obtained for Tbp by dividing the mean of
the triplicates for the test genes with the mean of the triplicates of
Tbp for each individual sample.
Standard Curves--
The four individual amplicons were
amplified from a liver cDNA preparation using standard PCR
methodologies, purified, and individually cloned into the PCR TOPO 2.1 cloning vector (Invitrogen, Netherlands). The cloned fragments were
sequenced to confirm that the insert was the correct sequence and
present in only a single copy. Purified plasmid DNA was prepared from
300-ml cultures using stages 1 to 4 of the Qiagen Plasmid Midi kit,
filtered, precipitated in absolute ethanol, washed in 70% ethanol,
resuspended in 1 ml of 10 mM Tris, 1 mM EDTA,
pH 8 buffer, and further purified by standard CsCl centrifugation and
dialysis. Plasmid concentrations were determined by spectrophotometry,
and purity was confirmed by agarose gel electrophoresis. Purified
clones were diluted and stored in single-use aliquots at Histology and Fiber Counts of Regenerating Muscle
Samples--
Muscles for histological analysis were mounted
transversely, and 8-µm thick cryostat sections were cut and stained
with hematoxylin and eosin. The number of fibers containing peripheral
or central nuclei was counted by light microscopy.
Statistics--
As the sample groups were small, comparisons
were performed using the non-parametric Mann-Whitney test. Data are
stated as showing a significant difference if p < 0.05.
Following initial optimization of the PCR protocols, all primer
pairs successfully amplified single products from cDNA, of the
expected sequence. No products were detected with genomic DNA template
or when excess no-RT controls were analyzed (data not shown),
demonstrating that the assays were specific for the transcribed
products. Analyses of different dilutions of selected cDNA
preparations on different days and using freshly made reaction mixes
showed that the variation between assays for individual samples did not
exceed 5%. The standard curves demonstrated linearity of the assays
with no change in the amplification efficiency over the tested range of
12 up to 1,200,000 copies of template.
Postnatal Expression of the DM1-locus Genes--
Fig.
1 summarizes the expression of the
DM1-locus genes in both neonatal and adult tissue samples.
Dmpk is the gene whose expression changes most markedly
during postnatal development, increasing in the male distal and
proximal skeletal muscles, the heart, and the cerebral cortex and
cerebellum. In females there is also an increase in Dmpk
expression in the distal skeletal muscles, the heart, and the small
intestine but no significant postnatal changes in the other tissues
(see "Discussion" for further information on the small intestine
data for the male mice). Postnatal up-regulation of Dmwd was
also observed. Dmwd was up-regulated in the male adult
distal skeletal muscles, cerebral cortex, and testis, but in contrast,
Dmwd levels decreased postnatally in the female cerebral
cortex. A similar postnatal decrease was seen in the female small
intestine. Six5 expression was down-regulated in adult male
proximal skeletal muscles and testis compared with the neonates, but
expression increased postnatally in the cerebral cortex. In female mice
the only significant change detected for Six5 was a
postnatal increase in expression in the small intestine.
Patterns of Expression of the DM1-locus Genes--
In Fig.
2 the graphs demonstrate the patterns of
DM1-locus gene expression in different mouse tissues. In certain
tissues Dmpk is strikingly much more highly expressed than
the other genes. In the distal and proximal skeletal muscles, the small
intestine, and the adult heart the levels of Dmpk
transcripts far exceed those of Dmwd and Six5 by
as much as 2 orders of magnitude. The Dmpk expression in
these tissues is also much higher than the levels of Dmpk in
the other tissues sampled, e.g. Dmpk is
approximately 100-fold more highly expressed in the adult female heart
than in the ovaries. It is also notable that in some tissues
Dmpk is not the most highly expressed of the genes. In
neonatal cortex and cerebellum, Dmwd is more highly
expressed that the other two genes, although this is less apparent in
the adult samples, except for the female cortex. Dmwd is
also the most highly expressed of the DM1-locus genes in the adult
testis. There is no tissue in which Six5 is the most highly
expressed of the three genes, and in the majority of samples it is
expressed at a very low level, particularly in the neonatal brain and
the adult testis. Due to technical constraints, we did not dissect out
the lens from neonatal mice and, therefore, do not have development
data for this tissue. However, it is apparent that all three DM1-locus
genes are expressed in the adult lens.
DM1-locus Gene Expression during in Vivo Skeletal Muscle
Regeneration--
Fig. 3 shows the
results obtained after in vivo regeneration of the tibialis
anterior muscle. Regenerated fibers are characterized by central
nuclei. 3 days post-injection the BaCl2-treated muscles consisted mainly of newly regenerated small diameter muscle fibers interspersed with connective tissue and some peripherally nucleated fibers that had not undergone regeneration. Counts of central nucleation were not recorded at 3 days because of the disparity in
fiber size and presence of connective tissue compared with the later
time points. 10 days post-injection treated muscles contained centrally
nucleated fibers (35-45%) of increased diameter, with minimal amounts
of surrounding connective tissue compared with the earlier time point.
A similar pattern was seen at 25 days post-injection (16-27% of
fibers centrally nucleated). Control contralateral muscles at all 3 time points consisted of close-packed muscle fibers of uniform diameter
with virtually no regeneration (data not shown). At 3 days
post-treatment Dmpk levels in the regenerating muscles were
significantly lower than in the controls. This was the only significant
difference seen in gene expression during in vivo
regeneration.
This report is the most extensive study addressing the issue of
postnatal expression patterns and developmental regulation of the DM1
locus genes and the only one to employ genuinely quantitative methodologies. The majority of previously published studies have analyzed expression of only one or, at most, two of the DM1-locus genes, making it very difficult to compare relative levels of gene
expression, e.g. to determine if in a particular tissue
Dmpk is more highly expressed than Dmwd or how
expression of a gene varies between tissues. In contrast, in this
report we have used the highly quantitative and reproducible technique
of fluorescent real-time RT-PCR, the most sensitive method of
quantification available for high throughput studies, which is rapidly
becoming the technique of choice in expression studies. Because of its extended linear detection range, the same technique can be used to
assess accurately transcript levels from genes that are expressed at
widely varying levels, an advantage not enjoyed by the methods previously applied to the DM1-locus genes. Additionally, by including a
standard curve for the relevant amplicon on every plate and analyzing
all genes for a tissue on a single diluted cDNA preparation, we
have abrogated the effects of any inter-test variation in amplification efficiency.
It is clear that there is a complex set of postnatal changes in
expression of the DM1-locus genes, and the genes are clearly independently regulated during postnatal development. The most striking
changes are seen for Dmpk. Expression of Dmpk
increases postnatally in skeletal muscles (with the possible exception
of distal muscles in females), the heart, and the small intestine in
females. It appears from Fig. 1 that similar increases occur in the
male small intestine, but we cannot perform statistical analyses as
n = 2 for the adult small intestine (despite repeated extractions and assays, the Tbp data for this sample failed
to match our selection criteria). It is noteworthy that these increased levels of Dmpk expression occur in tissues rich in either
striated or smooth muscle. The workload of the muscle cells in all
these tissues increases considerably during postnatal development, and it may be inferred that increased levels of Dmpk are
required under these conditions. Whether this increase is mediated
through mechanotransductive processes or is a consequence of changing hormonal influences remains to be established. Preliminary data from
juvenile mice (3 weeks old) suggested that in distal skeletal muscles
and hearts much of this up-regulation was established within the first
few weeks of birth (data not shown). The increased expression of
Dmpk in these samples does not represent merely a general
pattern in which expression of all non-housekeeping genes is
up-regulated, as it is noticeable that expression of Dmwd in
the small intestine falls dramatically postnatally.
Expression of the DM1-locus genes in the cerebral cortex also seems to
exhibit significant levels of postnatal regulation. In all male mice
there is significant up-regulation of each gene in the adult cortex
compared with neonates, but the same effect is not observed for female
mice, where levels of Dmwd drop significantly postnatally.
It is not known if these gender-specific differences represent a
physiologically relevant phenomenon, perhaps related to hormonal influences.
The postnatal expression of the DM1-locus genes does not alter
significantly in the ovaries, in contrast to the testis. Levels of
Dmwd are significantly increased in the adult testis
compared with the neonates, but the opposite trend is observed for
Six5. Dmpk levels in the testis do not change postnatally,
again reinforcing the concept that there is no obligatory co-ordinate
regulation of the genes at this locus.
As our technique is standardized, we can calculate definitively the
absolute expression patterns of the DM1-locus genes within and between
samples. In the skeletal muscles, heart, and small intestine,
expression of Dmpk far outstrips that of Dmwd or
Six5, in some cases by more than 2 orders of magnitude. In
the other tissues analyzed, there are fewer major differences between
the expression of the genes, with the exception of the cerebral cortex and adult testis, in which Dmwd is the most highly expressed
of the DM1-locus genes. It has been suggested for some time that Dmwd has its highest expression levels in these tissues
(19), but ours is the first report showing that these levels outstrip those of the other DM1-locus genes. In all adult tissues it was noticeable that Six5 was never the most highly expressed
gene, and in some tissues its expression was markedly reduced compared with Dmpk and Dmwd, although we cannot exclude
the possibility that there may be small subsets of cells overexpressing
a particular gene in a heterogeneous tissue sample. Cell-specific
expression can only be determined by in situ hybridization
or immuno-histochemistry if suitable antibodies become available, but
neither of these techniques is genuinely quantitative. The other
alternative, of micro-dissection of samples prior to the TaqMan
analyses, is not technically feasible for this work.
Our data also indicate that, unlike Dmwd and
Six5, expression of Dmpk is influenced by the
regenerative status of the muscle. Dmpk is down-regulated
during skeletal muscle regeneration, and taken together with the
previously published cell culture work (25, 26), this suggests that
very elevated levels of this transcript may be incompatible with the
fusion of myoblasts to the terminally differentiated myotube state.
This may introduce additional difficulties in analyzing DMPK
expression data from patients with DM1, as it will be important to
determine if apparent changes in levels of DMPK in skeletal
muscles between DM1/non-DM1 patients are actually a primary molecular
effect or a secondary consequence of increased regeneration in a
particular sample.
What are the implications of the data published here for an
understanding of the pathology observed in DM1? Probably the most clear-cut conclusions can be drawn from the data for the adult lens.
Winchester et al. (33) analyzed the expression of
SIX5 and DMPK in the human lens using in
situ hybridization and RT-PCR. They were unable to detect
transcripts for DMPK and concluded that SIX5 was
thus a much stronger candidate for involvement in the formation of the
characteristic cataracts that are such a common feature in this
disease. In contrast, transcripts for all three DM1-locus genes were
detected in our adult mouse lens samples. This is unlikely to reflect
contamination by other adherent tissues. Samples were collected by a
veterinarian with considerable experience of mouse anatomy, and every
effort was taken to minimize tissue contamination compatible with
minimizing the time before samples were frozen. It is also unlikely
that our data represent a simple cross-species difference. In our hands
transcripts for all three genes can be readily detected by RT-PCR in
freshly isolated human primary lens epithelial
cells,2 casting considerable
doubt on any model of cataract formation solely based around
SIX5.
DM1 has a very complex multisystemic phenotype and may be more
correctly described as a single-locus than a single-gene disorder, i.e. functional haploinsufficiency for one or more of the
genes may vary between tissues and ages, depending on the physiological threshold for each gene product. However, attempts to identify molecular pathological mechanisms in DM1 based on models of gene dosage
have been severely hampered by the lack of background data available on
the normal expression patterns of the DM1-locus genes. This paper
attempts to address this by providing the first genuinely quantitative
data that allows cross-comparisons between the different genes.
Although this work was carried out in a model organism rather than in
human material, it provides a useful starting point for generating a
developmental map of expression.
During mapping efforts to identify human disease genes, it is quite
common to be faced with a number of candidate genes from a defined
genetic interval. Under these circumstances, a common strategy is to
investigate which of the candidates shows the closest correlation
between the expression of the gene and the expression of the phenotype.
The same rationale can be applied to the DM1 locus, whereby we have
three candidate genes for which we need to determine if one of them
shows a close correlation between phenotype and expression.
Intriguingly, such a correlation appears to exist between the tissues
most commonly involved in adults with DM1 and Dmwd. Its
levels are highest in lens, testis, and heart and lowest in the tissues
rarely implicated, e.g. ovaries and liver (although DM1
patients are often insulin-resistant, this is a purely skeletal muscle
phenomenon, as the hepatic response to insulin is normal (34)). The
high expression in the lens may be significant, as this tissue acts as
the most sensitive indicator of DM1 pathology in minimal expansion
patients. Such a correlation is less apparent for the other genes,
e.g. Dmpk levels are lowest in the adult gonads,
cerebral cortex, cerebellum, and lens, all of which, with the exception
of the ovaries, are commonly implicated in DM1. Six5 levels
in adults are higher in the liver than the cerebral cortex or testis,
inconsistent with the DM1 phenotype. Dmwd levels are also
very low in the small intestine, but the pathological data on the small
intestine is difficult to interpret. Gastro-intestinal disturbances are
commonly reported in DM1 with abnormalities in the large bowel (35) and the duodenum (36) (not analyzed in our study) clearly demonstrated, but
more research is required to determine the extent to which the rest of
the small intestine is involved. Our data suggest this issue of the
precise patterns of tissue involvement in the phenotype may be
important. The apparent correlation of symptom presentation with
Dmwd expression is intriguing, but unfortunately, very few
molecular studies have analyzed the expression of Dmwd in
patient material. Although our own work suggests that the cytoplasmic levels of DMWD transcripts in skeletal muscle may fall
within those found in the control population (24), an allele-specific decrease in DMWD transcripts from the expanded allele has
been reported (22). The data here suggest that this gene may warrant closer examination than it has previously been awarded to establish conclusively if in a range of DM1 patient tissues, including individual muscle fibers, there is a genuine decrease in DMWD
transcripts. As there is currently no data on the activities and
intracellular functions of DMWD protein, it is difficult to speculate
at this stage on potential pathological pathways that might result if there is haploin sufficiency of this gene product in patient tissues.
In summary, our data have shown that in both muscle regeneration and
postnatal expression there is independent regulation of the three
DM1-locus genes. This has implications for developing molecular models
of DM1, including differentiating between mechanisms for congenital and
adult forms of the disorder. Although the very high level of expression
of Dmpk suggests it has an important role in adult skeletal
muscle function, it is down-regulated during muscle regeneration.
Additionally this research demonstrates that SIX5 is not the
sole candidate for cataractogenesis and that DMWD may be a
stronger candidate for involvement in many aspects of the adult
phenotype than has generally been recognized.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until
processed. The tissue samples were collected from neonatal and adult
(10 weeks of age) mice. In some cases, samples from juvenile mice (3 weeks of age) were also analyzed. 3 males and 3 females were dissected
at each time point.
165 °C in liquid nitrogen.
Rn value was calculated based on the equation,
Rn = Rn+ Rn
. Rn+ is
the ratio of reporter dye emission and passive reference dye emission
at any given point during the PCR reaction. Rn is the
ratio of reporter base-line emission and passive reference dye emission
during cycles 3-13. The fluorescence threshold was set within the
linear phase across all the samples in a particular run. The point at
which each individual sample Rn crossed the threshold was recorded
as its Ct value. This is linearly related to the log of the initial
amount of template in each reaction, allowing estimation of initial
number of copies in the unknown samples by comparison with standards.
20 °C,
and the same diluted preparations were used throughout.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
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Fig. 1.
Neonatal and adult expression of the
DM1-locus genes in C57Bl/10 mouse tissues. Tissue samples are
listed on the vertical axis. D, distal skeletal muscles;
P, proximal skeletal muscles; H, heart;
Co, brain cortex; Ce, brain cerebellum;
Li, liver; G, gonads; Si, small
intestine; Le, lens. Mean levels of expression of the
DM-locus genes, normalized to Tbp, are displayed
logarithmically on the horizontal axis. Panels
A-C, males; panels D-F, females.
Panels A and D, Dmpk;
panels B and E, Dmwd;
panels C and F, Six5. Empty
bars, neonatal; filled bars, adult. p < 0.05 is marked with an asterisk. For clarity, only
positive error bars (S.D.) are shown. For all categories,
n = 3 mice, except for adult male small intestine,
where n = 2 (see "Discussion").
View larger version (33K):
[in a new window]
Fig. 2.
Relative expression of the DM1-locus genes in
C57Bl/10 mouse tissues. Tissue samples are shown on the
horizontal axis. D, distal skeletal muscles;
P, proximal skeletal muscles; H, heart;
Co, brain cortex; Ce, brain cerebellum;
Li, liver; G, gonads; Si, small
intestine; Le, lens. Mean levels of expression of the
DM-locus genes, normalized to Tbp, are displayed
logarithmically on the vertical axis. A, neonatal males;
B, neonatal females; C, adult males;
D, adult females. Solid black bars,
Dmpk; empty bars, Dmwd; hatched
bars, Six5. For clarity, only positive error bars
(S.D.) are shown. For all categories, n = 3 mice,
except for adult male small intestine, where n = 2 (see
"Discussion").
View larger version (65K):
[in a new window]
Fig. 3.
Expression of the DM1-locus genes during
in vivo muscle regeneration. Mean levels of
expression of the DM-locus genes, normalized to Tbp, are
displayed logarithmically on the vertical axis for days 3, 10, and 25 post-treatment (A-C). On the horizontal axis,
the genes assayed are shown and the experimental conditions (+ denotes
BaCl2, denotes control). For clarity, only positive
error bars (S.D.) are shown, and n = 3. p < 0.05 is marked with an asterisk.
Hemeotoxylin and eosin sections of the BaCl2-treated
muscles are shown at the same time points. R, regenerating
areas; NR, non-regenerating. Scale
bar, 50 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
|---|
We thank Edward Schmidt for Tbp intron/exon boundaries, Ulrike Kronenewett for valuable discussion of the TaqMan assay, Louise Brown for statistical advice, Helen Aldridge for technical assistance with histology, and Martin Gosling for help with Fig. 3.
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FOOTNOTES |
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* This work was funded by the Muscular Dystrophy Campaign of Great Britain and Northern Ireland, Swedish Medical Research Council Grant 3875, the Swedish Association of Neurologically Disabled (NHR), the Petrus and Augusta Hedbergs Foundation, and the Torsten and Ragnar Söderberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 46 8 5177 3920; Fax: 46 8 5177 3620; E-mail: Maria.Eriksson@cmm.ki.se.
Published, JBC Papers in Press, March 30, 2000, DOI 10.1074/jbc.M001592200
2 N. Carey, manuscript in preparation.
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ABBREVIATIONS |
|---|
The abbreviations used are: DM1, myotonic dystrophy; RT-PCR, reverse transcription-polymerase chain reaction.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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