We have used gene targeting to create a mouse
model of glycogen storage disease type II, a disease in which distinct
clinical phenotypes present at different ages. As in the severe human
infantile disease (Pompe Syndrome), mice homozygous for disruption of
the acid
-glucosidase gene
(6neo/6neo) lack enzyme
activity and begin to accumulate glycogen in cardiac and skeletal
muscle lysosomes by 3 weeks of age, with a progressive increase
thereafter. By 3.5 weeks of age, these mice have markedly reduced
mobility and strength. They grow normally, however, reach adulthood,
remain fertile, and, as in the human adult disease, older mice
accumulate glycogen in the diaphragm. By 8-9 months of age animals
develop obvious muscle wasting and a weak, waddling gait. This model,
therefore, recapitulates critical features of both the infantile and
the adult forms of the disease at a pace suitable for the evaluation of
enzyme or gene replacement. In contrast, in a second model, mutant mice
with deletion of exon 6 (
6/
6), like the recently published acid
-glucosidase knockout with disruption of exon 13 (Bijvoet, A. G., van de Kamp, E. H., Kroos, M., Ding, J. H., Yang, B. Z., Visser, P., Bakker, C. E., Verbeet, M. P., Oostra,
B. A., Reuser, A. J. J., and van der Ploeg, A. T. (1998) Hum. Mol. Genet. 7, 53-62), have unimpaired
strength and mobility (up to 6.5 months of age) despite
indistinguishable biochemical and pathological changes. The genetic
background of the mouse strains appears to contribute to the
differences among the three models.
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INTRODUCTION |
In glycogen storage disease type II
(GSDII),1 an autosomal
recessive disorder, the failure of acid
-glucosidase (GAA, acid maltase, EC 3.2.1.20) to hydrolyze lysosomal glycogen leads to the
abnormal accumulation of large lysosomes filled with glycogen in some
tissues (2). The most severe form, Pompe Syndrome, is a rapidly
progressive disease in which heart failure is fatal in infancy. In
milder forms, there is progressive skeletal muscle weakness, and death
may result from pulmonary failure secondary to diaphragmatic weakness
as late as the seventh decade.
There is currently no effective therapy, but several candidate
therapies, based on the discovery that acid
-glucosidase, like many
other lysosomal enzymes, is secreted and can be taken up through cell
surface mannose-6-phosphate receptors on other cells (3-5), are
already under development (6-12). These studies stimulated efforts to
create a mouse model suitable for testing enzyme replacement and gene
therapies. Bijvoet et al. (1) recently reported the
generation of knockout mice which develop generalized glycogen storage
and cardiomegaly but remain phenotypically normal.
We describe here the generation of two models: 1) knockout mice in
which the GAA gene is disrupted by a neo insertion in exon 6 (6neo/6neo) and 2) mutant
mice in which exon 6 of the GAA gene and the neo gene are
removed by Cre/lox-mediated recombination (
6/
6) (13). In both models, animals develop biochemical and pathological changes similar to those in humans, but only
6neo/6neo mice show early
signs of reduced mobility and muscle strength. By 8-9 months of age
6neo/6neo mice develop a
weak, waddling gait, and progressive muscle wasting.
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EXPERIMENTAL PROCEDURES |
Construction of Targeting Vector, Transfection of Embryonic Stem
(ES) Cells, and Breeding--
GAA genomic clones were isolated from a
129/Sv mouse genomic library. A plasmid containing both the
neomycin-resistance (neo) gene and the herpes virus
thymidine kinase gene in the pBluescript vector (a gift of Dr. R. Proia) served as the backbone of the targeting vector (14). The
organization of the targeting construct is shown in Fig. 1A.
A genomic fragment extending from an XbaI site in intron 2 to a BamHI site in exon 6 was inserted into the XhoI site between the thymidine kinase and neo
genes. In addition, a termination codon and a new EcoRV site
were introduced within exon 6 upstream from the neo gene.
Next, a genomic fragment containing the remainder of exon 6 and exons 7 through 13 was cloned into the SalI site downstream of the
neo gene. Two loxP sites were inserted into
introns 5 and 6. The resulting vector has ~2.7 kb of homology
upstream and ~4.3 kb of homology downstream of the neo
gene. The linearized vector was electroporated into 129/Sv RW4 ES cells
(Genome Systems Inc.), and the resulting neo-positive (G418-resistant), thymidine kinase-negative (ganciclovir-resistant) clones were screened by Southern analysis. Chimeric mice were generated
by blastocyst injection of heterozygous ES cells into 3.5-day C57BL/6
embryos. Six independent cell lines containing the disrupted GAA allele
were used to make chimeras that were bred to C57BL/6 females to
generate heterozygous mice (F1). Four mutant lines were
then established through germ line transmission; heterozygous
F1 mice derived from two independent cell lines, 2-55 and
2-86, were intercrossed to obtain mice homozygous for the disrupted
allele (F2 and F3). Alternatively,
F1/2-55 and F1/2-86 heterozygous mice were bred
to EIIa-cre transgenic mice (FVB/N) for Cre-mediated
deletion (
6/
6) of the neo gene and exon 6 of the GAA
gene in vivo.
Enzyme Assay and Western Blot Analysis--
GAA activity in the
homogenates of skeletal muscle, liver, heart, and tail was measured as
conversion of the substrate
4-methylumbelliferyl-
-D-glucoside to the fluorescent
product umbelliferone as described previously (1, 15). Tissues were
dissected and homogenized in lysis buffer (300 mM NaCl, 50 mM Tris, 2 mM EDTA, 0.5% Triton X-100) with
proteinase inhibitors (4 mM Pefabloc SC, 10 µg/ml
aprotinin, 10 µg/ml leupeptin). Samples (50 µg protein) were
electrophoresed on 10% SDS-polyacrylamide gel electrophoresis gels and
electrotransferred to nitrocellulose membranes. The blots were blocked
with bovine serum albumin and incubated with rabbit antiserum to human
placental GAA or rabbit antiserum to human urine GAA (kindly provided
by Dr. F. Martiniuk and Dr. A. J. J. Reuser). Immunodetection
was performed with goat anti-rabbit IgG conjugated to horseradish peroxidase in combination with chemiluminescence (ECL, Amersham Life
Science Inc.).
Isolation of RNA and DNA, cDNA Synthesis, RT-PCR, and
Southern Analysis--
RNA was isolated from skeletal muscle and liver
using a Total RNA Kit (Qiagen). First strand cDNA synthesis was
primed from 2 µg of total RNA with 50 ng of random hexamers according
to the manufacturer's instructions (Boehringer Mannheim). Two µl of
the cDNA sample were used as a template for PCR amplification with primers flanking the neo gene: cctttctacctggcactggaggac
(exon 5 sense) and ggacaatggcggtcgaggagta (exon 7 antisense) or
tcaccctctggaaccgggacacacca (exon 4 sense) and
ccggccatcctggtgcagctcccgca (exon 8 antisense). The second set of
primers was used to detect any possible transcripts in which the
neo gene may be spliced out. PCR reactions were carried out
for 35 cycles that consisted of 50-s denaturation at 95 °C, 50-s
annealing at 55 °C, and 2-min extension at 72 °C using PCR SuperMix (Life Technologies, Inc.). Genomic DNA isolated from ES cells
or mouse tails was digested with EcoRV, electrophoresed on
1% agarose gels, and transferred to Nytran membranes. The
hybridization probe was generated by PCR, and labeled by the random
hexamer method after gel purification.
Histology--
For electron microscopy, tissues were fixed in
phosphate-buffered saline containing 4% formaldehyde and 2%
glutaraldehyde followed by post-fixation in 1% osmium in 0.1 M cacodylate buffer. The tissues were rinsed in an aqueous
solution containing 4.5% sucrose, dehydrated in a series of graded
alcohol solutions, rinsed in 100% propylene oxide, and embedded in
epoxy resin. Thin sections (60 to 70 nm) were double-stained with
uranyl acetate and lead citrate. The stained sections were stabilized
by carbon evaporation and photographed with a Hitachi H7000 electron
microscope operated at 75 kV. For light microscopy, sections from
tissues were fixed in 10% formalin, processed, embedded in paraffin,
and stained with hematoxylin-eosin or periodic acid-Schiff (PAS) by
standard methods.
Behavioral Testing--
All procedures were conducted in
accordance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals. Locomotor activity in an open field was
measured in a Digiscan apparatus (model RXYZCM, Omnitech Electronics).
Total distance, horizontal activity, and vertical activity were
measured by the total number of photocell beam breaks in 10- or 15-min intervals over 1 h, and data averaged over these periods were used
for analysis. Three to six independent testing sessions were conducted
for each group over a period of 1-2 weeks. Male mice were tested at
ages 3.5-6, 8-9, and 10.5-22 weeks. Fourteen
6neo/+, 11 6neo/6neo, 8
6/+, and
9
6/
6 mice were used for the test. The origin of the mice which
were phenotypically tested is indicated in Table I. Statistical analyses were performed
using the one-way analysis of variance test (Sigmastat program). The
ability to hang upside down from a wire screen placed 60 cm above a
large housing cage was measured as latency to fall into the cage.
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RESULTS |
The murine GAA gene was disrupted by insertion of neo
into exon 6, with the expectation that the disruption would completely block gene expression
(6neo/6neo). In addition,
loxP sites were placed in the introns flanking the disrupted
exon 6 so that exon 6 could be precisely removed (
6) by mating to
Cre-producing mice. In humans, a similar splicing mutation around exon
6 is associated with a relatively mild phenotype (16).
Generation of Mice with Exon 6 Disruption of the GAA Gene--
By
homologous recombination in ES cells, we created a mutant GAA allele in
which a neo cassette disrupts the gene within exon 6 (Fig.
1A). A termination codon and a
new EcoRV site were introduced into exon 6 upstream from the
neo gene. Translational termination at the stop codon in
exon 6 would result in the synthesis of a truncated protein of ~36
kDa. The frequency of recombination was 1 in 4 G418/ganciclovir-resistant clones. Recombinant clones were used to
produce chimeric mice that transmitted the mutation through the germ
line. Heterozygous mice (F1) derived from two independent cell lines (2-55 and 2-86) carrying the targeted allele were used for
further breeding. Genotyping of the mice generated by intercrossing of
heterozygotes (Fig. 1B) revealed the expected Mendelian
ratio, indicating no effect on embryonic development.

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Fig. 1.
Disruption of the GAA gene in mouse ES cells,
germ line transmission of the disrupted allele, and expression of
murine GAA in wild-type and 6neo/6neo mice.
A, structure of the targeting vector (middle) and
a partial restriction map of the GAA locus before (upper)
and after (lower) homologous recombination. A neo
cassette was inserted in exon 6 of the gene. A ~300-bp probe in
intron 2 (external to the targeting construct) detects a ~6-kb
fragment from wild-type DNA and a mutant specific fragment of ~2.7
kb. Restriction sites are: B, BamHI;
RI, EcoRI; RV, EcoRV;
H, HindIII; X, XbaI.
B, initial screen of the targeted GAA clones by Southern
blot hybridization analysis (left). The EcoRV
2.7-kb fragments indicated the expected recombinant allele. Southern
blot analysis of tail DNA from wild-type (+/+), heterozygous (+/ ),
and homozygous ( / ) offspring in the F2 generation
derived by crossing F1 carriers for the GAA disruption
(right). C, RT-PCR analysis of muscle cDNA
from wild-type (+/+), heterozygous (+/ ), and homozygous ( / ) mice.
The primers in exons 5 and 7 flanking the neo gene detected
a 265-bp amplification product in the wild-type (+/+) and heterozygous
(+/ ) but not in homozygous ( / ) mice (top). Similarly,
the primers in exons 4 and 8 detect a 478-bp product only in the
wild-type (+/+) and heterozygous (+/ ) mice (bottom). The
RT-PCR negative control (NC) was carried out by omitting RNA
from reverse-transcription reaction. M, DNA marker.
D, Western analysis (shown for liver). The blot was probed
with rabbit IgG to human urine GAA (I) or human placental
GAA (II).
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Reverse transcription-PCR with two sets of primers flanking the
neo gene detected wild-type products in the wild-type (+/+) and heterozygous (+/
) but not in
6neo/6neo (
/
) mice
(Fig. 1C), indicating that normal mRNA is not made in
homozygotes. However, mRNA amplification with primers in exon 12 (sense) and exon 14 (antisense) downstream from the neo gene detected a low level of transcripts in
6neo/6neo mice (not
shown). Similarly, RT-PCR with primers in exon 5 (sense) and the
neo gene (antisense) detected a very low abundance message in the 6neo/6neo;
reamplification of the PCR product with nested primers followed by
sequencing established that the termination codon introduced into exon
6 upstream from the neo gene remained intact (not
shown).
In homozygous mice, no GAA protein was detected by Western analysis
(Fig. 1D) using antibodies against either human urine or
human placental GAA. The absence of functional protein in
6neo/6neo mice was
confirmed by enzyme assay in the lysates of multiple tissues (Table
II). The residual levels of enzyme
activity (at the standard pH 4.3) in the muscle, heart, and tail
samples of 6neo/6neo mice
did not exceed the background level found in a fibroblast cell line
from an infantile patient (0.64 nmol of
4-methylumbelliferyl-
-D-glucoside/h/mg of protein; cell
line 4912) in which mRNA is not expressed (17). At low pH 3.6 (1)
the enzyme activity was below the detection limits (0.7-1.0 ng of
4-methylumbelliferone/10-µl reaction).
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Table II
GAA activity (nmol of
4-methylumbelliferyl- -D-glucoside/h/mg of protein) in
tissues and tail samples
GAA activity was measured under standard conditions, pH 4.3 (15).
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6neo/6neo Mice Accumulate Glycogen in
Lysosomes--
Abnormal lysosomal glycogen storage was found in the
heart and skeletal muscle of
6neo/6neo mice. Electron
microscopy showed the progressive accumulation of membrane-limited
organelles between the bundles of myofibrils at the earliest point
examined - age 3 weeks (Fig. 2
a and d). Immunoelectron microscopy with an
antibody specific for the LAMP-1 protein confirmed that the organelles
were lysosomes (not shown). Over time, the lysosomes increased in size
and number (Fig. 2, b, c, e, and
f). Furthermore, the density of the accumulated glycogen particles within the lysosomes increased (Fig. 2, h and
i). The accumulation was clearly more marked in the heart
than in the skeletal muscle (Fig. 2, d-f and
a-c). Importantly, in the
6neo/6neo mice, there is
a significant reduction in the number of myofibrils, loss of lateral
myofibrillar registration, and signs of sacromere degradation,
especially the deformation at the Z lines. Some lysosomes appear broken, suggesting that the leakage of lysosomal proteases may
have contributed to the damage of the muscle structure.

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Fig. 2.
Electron microscopy of heart and skeletal
muscle in 6neo/6neo mice at different stages.
Top panel, skeletal muscle, a,
b, and c at 3, 10, and 15 weeks,
respectively (× 9,000). Middle panel, heart, d,
e, and f at 3, 10, and 15 weeks,
respectively (× 9,000). Bottom panel, muscle, g
at 15 weeks (× 30,000); heart, h and i at 3 and
15 weeks, respectively (× 30,000). Note different degree of glycogen
density in heart lysosomes at 3 and 15 weeks; at 15 weeks, all
lysosomes are densely packed with glycogen.
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Light microscopy (at 8 weeks) showed PAS-positive, diastase-sensitive
material in vacuoles in the heart and skeletal muscle (Fig.
3, b and d). In
animals examined at 14 weeks, the diaphragm showed PAS-positive
vacuoles by light microscopy (Fig. 3f).

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Fig. 3.
Analysis by light microscopy of heart,
skeletal muscle, and diaphragm in 6neo/6neo mice.
PAS-stained sections of (a and b) skeletal muscle
at 8 weeks of age (× 300 and 750, respectively); (c and
d) heart at 8 weeks of age (× 750); and (e and
f) diaphragm at 14 weeks of age (× 1000).
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6neo/6neo Mice Display Significant
Behavioral Abnormalities--
Although the mutant mice appeared
normal, when placed in an open field environment
6neo/6neo mice
consistently performed significantly worse than heterozygous littermates by several measures of locomotion (Fig.
4). Reduced activity was registered as
early as 3.5 weeks of age and was particularly striking for vertical
motion (Fig. 4, bottom panel). Similarly, in the wire-hang
task, which measures muscular function and grip strength,
6neo/+ mice outperformed
6neo/6neo littermates. At
15-16 weeks of age,
6neo/6neo mice were
almost never able to hold on to the inverted screen for more than 2 min
(once in 12 tests), whereas in 8 of 12 tests 6neo/+ littermates were able to remain hanging
for more than 2 min, and 4 of 12 heterozygous littermates were still
holding on at 5 min when the test was stopped. Older mice (8-9 months
of age) show obvious signs of muscle weakness with a weak, waddling
gait and muscle wasting (Fig. 5).
Offspring from independent mutant mouse lines were phenotypically
indistinguishable.

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Fig. 4.
Locomotor activity of 6neo/+ and
6neo/6neo mice. Top panel,
mean (±S.E.) horizontal activity per min in the open field (measured
by the number of photocell beam breaks). Middle panel, mean
(±S.E.) total distance (cm) per min in the open field. Bottom
panel, mean (±S.E.) vertical activity per min in the open field
(measured by the number of photocell beam breaks). A
6neo/6neo and a
6neo/+ animal were tested simultaneously. Each
bar represents the performance of two to four animals, and
no less than 44 intervals were averaged for each bar. Asterisks (***)
indicate performance significantly different from
6neo/+ mice at the 0.0001 level.
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Fig. 5.
Clinical signs of muscle weakness. This
8-month-old female
6neo/6neo mouse has
wasted lower back muscle and displays its hind limbs in a splayed
posture.
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Generation and Characterization of Mice with an In-frame Deletion
of Exon 6 of the GAA Gene (
6/
6)--
In the second model, the
disrupted exon 6 of the GAA gene and the neo gene were
totally excised from early embryos by breeding 6neo/+ mice (F1/2-55 and
F1/2-86; 129/C57BL/6 background) to transgenic homozygous
EIIa-cre (18) mice (FVB/N background) in which the adenovirus promoter confines the expression of Cre to an early stage of
pre-implantation development. F1 heterozygous (mouse lines
2-55/cre and 2-86/cre) for exon 6-deleted allele
were subsequently intercrossed to obtain F2 and
F3 homozygous mice (
6/
6). Cre-mediated deletion was
detected by PCR with primers in exon 5 and exon 7 (Fig.
6). As expected, the genomic sequence in
homozygous mice contained the 5' part of intron 5, then a single
loxP site in place of exon 6, followed by the 3' part of
intron 7 and exon 7 of the gene (not shown). RT-PCR with two sets of
primers (in exons 5/7 and exons 4/8) showed that the mutant mRNA is
produced (Fig. 7A), and that
in this mRNA exon 5 is spliced to exon 7, resulting in a precise
in-frame deletion of exon 6 (not shown).

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Fig. 6.
Detection of Cre-mediated deletion
( 6/ 6). A, the structure of the targeted GAA locus
before (upper) and after (lower) in
vivo Cre-mediated deletion. The two arrowheads
represent the loxP sites. B, the primers in
exon 5 (sense) and exon 7 (antisense) detect 687- and 505-bp PCR
fragments corresponding to the wild-type and exon 6 deleted alleles,
respectively (left panel). Genotype analysis of the
offspring from a 6neo/+ × EIIa-cre
cross (right panel). PCR analysis of tail DNA in the
F1 (top) and F2 (bottom)
generations. Both the 687- and 505-bp bands were detected in
heterozygotes (+/ ); only the larger band is seen in the wild-type
(+/+), and only the smaller band in homozygous ( / ) mice.
M, DNA marker; NC, negative control.
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Fig. 7.
Expression of the exon 6-deleted allele.
A, RT-PCR analysis of muscle cDNA from 6/ 6 mice.
Primers in exons 4 and 8 detect a 354-bp amplification product; primers
in exons 5 and 7 detect a 150-bp product. The sizes of the products
correspond to those expected for mRNA with exon 6 deleted. Each PCR
was done in duplicate. The RT-PCR negative control (NC) was
carried out by omitting RNA from reverse-transcription reaction in
which both sets of primers were used. M, DNA marker.
B, Western analysis (shown for liver). The blot was
probed with rabbit IgG to human placental GAA. Lane 1,
6neo/+; lane 2,
6neo/6neo; lane
3, 6/ 6.
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The
6/
6 mice were similar to the
6neo/6neo animals with
respect to the level of enzyme activity measured in tail skin, muscle, and liver (not shown), absence of protein (Fig. 7B), and
accumulation of lysosomal glycogen in skeletal muscle, heart, and
diaphragm (Fig. 8). Strikingly, however,
unlike the 6neo/6neo
mice, their performance in the open field was similar to that of
heterozygous
6/+ littermates derived from two mouse lines (Fig.
9, Table I). Interestingly, in all
measures of activity, the
6/+ mice outperformed the
6neo/+ animals, indicating a genetic difference
between the two mouse strains. So far (up to 6.5 months of age) the
6/
6 mice have not developed any clinical symptoms.

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Fig. 8.
Analysis by light microscopy of skeletal
muscle, heart, and diaphragm in 6/ 6 mice. PAS-stained
sections of a, skeletal muscle at 8 weeks of age (× 750);
b, heart at 8 weeks of age (× 750); and c,
diaphragm at 11 weeks of age (× 1000).
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Fig. 9.
Locomotor activity of mice with exon 6 deletion (heterozygotes, 6/+; homozygotes, 6/ 6), and exon 6 disruption (heterozygotes, 6neo/+; homozygotes,
6neo/6neo). Top panel, mean (±S.E.)
horizontal activity per min in the open field (measured by the number
of photocell beam breaks). Middle panel, mean (±S.E.) total
distance (cm) per min in the open field. Bottom panel, mean
(±S.E.) vertical activity per min in the open field (measured by the
number of photocell beam breaks). Each bar represents the
performance of 8-14 animals, and ~200 intervals (10 min each) were
averaged for each bar.
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DISCUSSION |
We have used an efficient method for generating two allelic
mutations at the murine GAA locus. The approach required the production of only one targeted mouse line with an exon 6 disrupted allele, which
served as a progenitor of the second line with exon 6 deleted allele.
Since the targeted locus contains two loxP sites flanking exon 6, the removal of the exon was performed simply by mating to
transgenic mice carrying Cre recombinase. The two models were designed
to replicate a range of clinical phenotypes:
6neo/6neo mice for a
severe phenotype, and
6/
6 mice for a milder disease. A milder
phenotype was predicted in the
6/
6 mice since a similar, though
not identical defect in a patient, splicing out exon 6 and the
inclusion of 7 new amino acids encoded by 21 nucleotides from IVS6,
resulted in 5-7% of residual enzyme activity and a juvenile form of
the illness (16). Both mouse models, however, resulted in apparently
complete "knockout," as shown by the virtual absence of enzyme
activity and the absence of GAA protein.
In humans, the severity and the age of onset of GSDII appear to depend
largely on the level of residual activity of the enzyme. Lack of enzyme
activity or extremely low levels (
1-2%) are associated with a fatal
infantile cardiomyopathy, whereas levels of 10-20% are associated
with an adult onset indolent skeletal myopathy (19-21). Unlike humans,
recently described knockout mice (9 months old) (1) and the
6/
6
mutants (6.5 months old) described here do not show clinical signs
despite a severe enzyme deficiency.
In contrast, 6neo/6neo
mice develop a progressive muscle weakness detectable as early as 3.5 weeks of age. The pathologic findings in
6neo/6neo mice indicate
accumulation of lysosomal glycogen in the skeletal muscle and
diaphragm, as in the adult human disease, and an even greater
accumulation in heart, a hallmark of infantile disease. Tests of
cardiac function will allow determination of the effects of the
glycogen accumulation in the heart. In quantitative tests of mobility
and strength, 6neo/6neo
mice moved less, especially in the vertical direction, and could not
hold on to a wire screen nearly as long as
6neo/+ littermates. By 8-9 months, clinical
signs of muscle weakness and muscle wasting are obvious. Longer
observation will be necessary to determine if this reduced strength
affects lifespan and if glycogen accumulation in the diaphragm reduces
lung function.
Thus, the 6neo/6neo model
has features of both the adult and the infantile forms of the human
disease, but the effects are attenuated. This difference in severity
and in pace between mice and humans is not surprising since the factors
which promote lysosomal glycogen storage are largely obscure. In
humans, for example, the deposition of glycogen is very different from
tissue to tissue within the same patient and from patient to patient or
even sibling to sibling although they may bear the same
mutation(s).
Of related interest in that regard are the observations that although
both the 6neo/6neo and
6/
6 mice have negligible enzyme activity and accumulate glycogen
in skeletal and heart muscles, the
6neo/6neo are weak in
open field and wire hang testing, but the
6/
6 are not. The
phenotypic difference between the two models described here cannot be
explained by the presence of a neo gene in the targeted
locus of the 6neo/6neo
mice: in both the phenotypically affected
6neo/6neo model and a
recently published phenotypically normal model with insertion of a
neo gene in exon 13, a hybrid GAA-neo mRNA
was detected by RT-PCR. Furthermore, we have studied the expression of
the neo gene in a mouse strain with disruption of the HexA gene which is known to perform normally in the open field (14). In this
strain, abundant neo transcripts were detected in both liver
and muscle by RT-PCR and sequencing (not shown), thus further indicating no effect of neo phosphotransferase on mobility
and muscle strength.
It is possible that the accumulation of glycogen is different in the
muscles crucial for the activities tested; or that accumulation of
glycogen in other sites such as the nervous system differs in the two
models; or that weakness is related not only to the amount of
accumulated glycogen. Indeed, the structural changes in myofibrillar
structure may relate to other factors besides simple glycogen
accumulation which are involved in lysosomal integrity. In support of
the last possibility, it should be noted that the two strains are of
different genetic background since the creation of the
6/
6
required mating to a strain bearing the Cre recombinase (FVB/N) while
the 6neo/6neo mice were
bred onto a C57BL/6 background. There is abundant similar evidence
illustrating the importance of genetic background and modifying genes
on phenotypic variation in knockout mice (22, 23). As shown in Fig. 9,
the background activity of the Cre strain control mice is substantially
greater than that of the controls for the
6neo/6neo mice,
suggesting that other genes influence behavior in the tests, and only
in the less active strain is the additional insult of glycogen
accumulation reflected in poorer performance. Such strain differences
may account for the apparent absence of weakness in the recently
published model (1), which, like the
6/
6 model described here,
was created on the 129/FVB background.
It should be noted that the level of residual activity in the exon 13 model was somewhat higher (2.6% in muscle and 3.8% in heart) than the
levels in the 6neo/6neo
and the
6/
6 mice when measured at the same low pH 3.6. At that low pH, neither of the knockout strains described here had detectable activity in tail skin, muscle, or heart. It is possible that a residual
low level of enzyme activity contributes to rescue of the phenotype of
the exon 13 published knockout (1). Longer observation of the
6/
6
mice (oldest animals are 6.5 months of age) may clarify this point.
Although all of the models created so far could be used for testing
proposed gene therapy or enzyme replacement since the pathological and
biochemical changes closely resemble those in humans, the
nondestructive and easily testable phenotypic abnormalities of the
6neo/6neo model suggest
that it would be the preferable choice.