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J. Biol. Chem., Vol. 276, Issue 38, 35995-35999, September 21, 2001
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§,
,,,,, and
From the Center for Immunology, The University of
Texas Southwestern Medical Center, Dallas Texas 75390-9093, the
¶ Department of Pediatrics and §§ Medical
Genetics, Tohoku University School of Medicine, 1-1, Seiryo-machi,
Aoba-ku, Sendai-city, Miyagi 980-8574, Japan, the Department of
Pathology and ¶¶ Institute of Gastroenterology, Tokyo
Women's Medical University, 8-1, Kawata-cho, Shinjuku-ku, Tokyo
162-8666, Japan, the ** Department of Pediatrics, Fukui Medical
University School of Medicine, 23, Shimoaizuki, Matsuoka, Fukui
910-1193, Japan, the Department of
Pediatrics, Shimane Medical University, 89-1 Enya-cho Izumo, Shimane
693-8501, Japan, and The Basel Institute for
Immunology, Grenzacherstrasse 487, CH-4005, Basel, Switzerland
Received for publication, June 13, 2001, and in revised form, July 16, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Propionic acidemia (PA) is an inborn error of
metabolism caused by the genetic deficiency of propionyl-CoA
carboxylase (PCC). By disrupting the Propionic acidemia (PA) is the most frequent autosomal recessive
disorder of organic acid metabolism in humans. It is caused by a
deficiency of propionyl-CoA carboxylase (PCC), a ubiquitously expressed, heteropolymeric mitochondrial enzyme involved primarily in
the catabolism of branched-chain amino acids and fatty acids, which
harbor odd-numbered chain lengths (1). A variety of mutations in the
genes of either subunit (PCCA, PCCB) abolish or reduce the PCC
function, resulting in a considerable clinical heterogeneity; some
patients die in early childhood due to rapid and furious acceleration
of the ketoacidosis in proportion to the increase of protein intake (2,
3). The primary treatment of PA has been dietary restriction of the
precursor amino acids of propionyl-CoA. Despite improvement of the
dietary therapy, however, its long-term efficacy still appears
controversial, and the overall outcome of severe forms of PA often
remains disappointing (1, 4, 5). As in other genetic disorders,
supplementation of PCC activity, perhaps primarily via gene therapy,
may efficiently temper even the severe forms of PA (6-9). It seemed
important, then, to generate an animal model of PA to test possible
therapeutic strategies. We therefore created a strain of mice lacking
PCC activity by disrupting the Generation of PCCA Generation of SAP+PCCA PCC Activity Assay--
PCC activity in the liver tissues was
determined by fixation of [14C]bicarbonate as described
elsewhere (12).
Histology--
Liver and kidney specimens were fixed in 10%
formalin. 10 µm-thick sections were cut from paraffin-embedded
samples and stained by hematoxylin-eosin or periodic acid-Schiff (PAS).
For fat detection, frozen liver sections, which had been embedded in
Tissue-Tek OCT compound, were cut at 10-µm thickness and then stained
with oil red O (Sigma) as described elsewhere (13).
Lethal Ketoacidosis in PCC-deficient Mice--
We created
PCC-deficient mice by disrupting the gene encoding the
Twenty-four h after birth, PCCA
As a consequence of progressed acidosis, PCCA
In the histology of tissues at 24 h after birth,
PCCA+/+ mice harbored significant amounts of a PAS-positive
product in the liver (Fig.
2A), which was identified as
glycogen by a glycogen-specific tissue staining method using
ethanol-fixed liver specimens (Fig. 2C). In contrast,
PCCA Fat Deposition in the Liver in PCCA Prevention of Lethal Ketoacidosis in Early Infant
PCCA
Three lines of SAP-PCCA transgenic mice were generated and
crossbred to PCCA
Fig. 3 demonstrates the RNA expression of
the transgene and the PCC activity in the liver as well as the
concentrations of the serum propionyl-carnitine and the urine 3-HP in
SAP+PCCA
This low PCCA transgene expression in
SAP+PCCA
The glycogen storage in the liver, which seemed to be consumed rapidly
in PCCA
Fat deposition in the liver was apparently decreased in
SAP+PCCA Requirement of a Larger Amount of PCC Supplementation after the
Late Infant Period--
Interestingly, the third line of
SAP+PCCA In this report, we established a mouse model that resembles
newborn PA patients who have undergone PCC gene transfer in a liver-specific fashion. These mice,
SAP+PCCA Based on these results of SAP+PCCA It may be worth emphasizing that SAP+PCCA In summary, PCCA
-subunit gene of PCC, we
created a mouse model of PA (PCCA
/), which died
in 24-36 h after birth due to accelerated ketoacidosis. A postnatal,
liver-specific PCC expression via a transgene in a far lower level than
that in wild-type liver, allowed PCCA/ mice to survive
the newborn and early infant periods, preventing a lethal fit of
ketoacidosis (SAP+PCCA/ mice).
Interestingly, SAP+PCCA/ mice, in which the
transgene expression increased after the late infant period, continued
to grow normally while mice harboring a persistent low level of PCC
died in the late infant period due to severe ketoacidosis, clearly
suggesting the requirement of increased PCC supplementation in
proportion to the animal growth. Based on these results, we propose a
two-step strategy to achieve an efficient PA prevention in human
patients: a partial PCC supplementation in the liver during the newborn
and early infant periods, followed by a larger amount of
supplementation in the late infant period.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit gene (PCCA
gene) via homologous recombination in embryonic stem
cells. To address the potentiality of a tissue-specific gene
supplementation as a PA therapy, we also generated a transgenic mouse
strain expressing PCCA exclusively in the liver and then crossbred
these two types of mice. By analyzing these mice, we determined the
potential therapeutic efficacy of the liver-specific PCC
supplementation and propose a new mode of treatment to achieve an
efficient PA prevention in human patients.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ Mice--
A mouse
genomic DNA clone containing exons of the PCCA gene was
isolated by screening a genomic DNA library derived from the 129/sv
strain. The targeting vector was prepared using the 12-kb EcoRI-HindIII genomic DNA fragment, pMC-1-neo-poly(A)
(Stratagene) plasmid and the pBluescript plasmid (Stratagene). This
construct was designed to delete a 2-kb
EcoRI-XbaI fragment from PCCA, encompassing exons
1-4 as schematized in Fig. 1A. The linearized targeting vector was electroporated into the E14.1 embryonic stem line as we
previously described (10). G418-resistant clones were isolated and screened by polymerase chain reaction and Southern blotting. Two of
146 Gly-418-resistant clones had undergone the desired homologous
recombination. Positive clones were injected into C57BL/6 (B6)
blastocysts, and male chimeric offspring were mated to B6 females. Mice
carrying the mutation in the heterozygous state (PCCA+/
) were
intercrossed to produce homozygote mutants (PCCA/).
/
Mice--
A cDNA clone containing the whole mouse PCCA coding
sequence was isolated by screening a mouse spleen cDNA
library (CLONTECH) using a human PCCA cDNA
fragment as a probe. The 2.8-kb mouse PCCA cDNA fragment was
subcloned into the EcoRI site of the pSG-1 expression vector, which
contains human SAP promoter and rabbit 
-globin non-coding
exon/intron (11). The complete plasmid (SAP-PCCA) was digested with
HindIII and XhoI, and the transgene fragment no
longer containing vector sequence was purified by Geneclean II kit
(Bio101, Vista, CA). DNA was microinjected into fertilized eggs of
(B6xDBA/2)F1 mice. Resulting founders were screened for transgene by
polymerase chain reaction and Southern blotting. Three independent
founders carried the transgene. All three mice were crossbred
with PCCA+/ mice. Progenies harboring
Transgene+PCCA+/ genotype were intercrossed
to obtain SAP-Transgene+PCCA/
(SAP+PCCA/) mice.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of
PCC protein (PCCA). Null mutation of the PCCA gene was
generated by replacing an ~2-kb fragment of the PCCA gene,
which contains exons encoding the N-terminal sequence of PCCA protein,
with the neomycin resistance gene (neor),
via homologous recombination in embryonic stem cells (Fig. 1A). Although the
PCCA gene is expressed ubiquitously from the early embryonic
stage throughout the whole
development,2
PCCA-null (PCCA/) mice were born at the expected
Mendelian frequency, and were indistinguishable from wild-type
(PCCA+/+) littermate mice at birth. This is compatible with
human PA patients, most of whom have no clinical complaints at birth
(1). During embryogenesis, the toxic propionic acid derived from
propionyl-CoA that has accumulated within cells might be purified via
maternal circulation. The abolished expression of PCCA in
PCCA/ mice was confirmed by Northern blot analysis of
the liver RNA (Fig. 1B), and by the absence of PCC enzyme
activity in the liver tissue (1590 ± 18 in PCCA+/+
versus 15 ± 17 in PCCA/, mice;
n = 3 for each, pmol/min/mg) (12).
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Fig. 1.
Disruption of PCCA
gene. A, knockout strategy. Partial restriction
maps are shown for the wild-type PCCA gene locus
(top), targeting vector (middle), and recombinant
gene locus (bottom). Exon positions were determined by
hybridization with short oligonucleotide primers and partial
sequencing. Most of exons 1-4 are located in a 2-kb
XbaI-EcoRI fragment but have not been mapped
accurately within this fragment (top). Exons, white
boxes. neor, neomycin resistance
gene (black box). Restriction sites: H,
HindIII; RI, EcoRI; Xb,
XbaI; Xh, XhoI. B, Northern
blotting. Fifteen µg of total liver RNA either from
PCCA / (left lane) or PCCA+/+
(right lane) mouse was hybridized with either PCCA or
-actin cDNA fragment. The same RNA filter was hybridized with
each probe after stripping the previous probe. No PCCA mRNA (~2.9
kb) was detected in PCCA/.
/ mice showed remarkably
high concentrations of serum propionyl-carnitine, urinary
3-OH-propionate (3-HP) and urinary methylcitrate, all of which increase
in human PA patients as the result of abnormal metabolism of the
accumulated propionyl-CoA, and thus are useful for PA diagnosis (1, 15) (Table I). Interestingly, the serum
propionyl-carnitine concentration was significantly high in
PCCA/ mice even at birth (Table I), suggesting that the
maternal circulation appears not to be able to completely overcome the
accumulation of the catabolites of propionyl-CoA. Probably due to the
intake of protein from the milk, as well as to the lack of the maternal circulation, the concentrations of propionyl-carnitine, methylcitrate, and 3-HP were strikingly increased in PCCA/ mice within
10 h after birth (Table I). The ketonuria, which indicates the
presence of acidosis caused by the catabolism of the accumulated
propionyl-CoA (1, 16), was detected 10 h after birth and was
rapidly accelerated in PCCA/ mice (Table I).
Rapid increase of abnormal metabolites in PCCA/ mice
/ (/) or PCCA+/+ (+/+) mice at various ages
on a filter paper (Guthrie card). Each number is the average of the
results of 4-6 mice. Urine 3-HP and MC were analyzed by the selected
ion monitoring (SIM) method (36). Urine ketone was assessed by a
test-tape (Lab-sticks, Bayer Co. Ltd). / mice at 24 h after
birth showed the maximum level (++++). N.D., not determined.
/ mice no
longer took milk after 10-12 h, although both PCCA/
and PCCA+/+ mice started sucking milk right after birth.
This decrease of intake is also often observed in human PA infants as a
primary symptom of the disease (1). Twenty-four h after birth,
PCCA/ mice were apparently dehydrated (dry skin, poor
urination), poor in movements, and possessed little or no milk in their
stomachs, whereas PCCA+/+ littermate mice were healthy and
their stomachs were filled with milk. A vicious circle by the
acceleration of dehydration and the progression of acidosis resulted in
death of all PCCA/ mice in 24-36 h after birth.
/ mice showed no or very little glycogen in the
liver (Fig. 2B). Interestingly, PCCA/ mice
did possess glycogen in the liver like PCCA+/+ mice at the
very late gestation, E18.5 (Fig. 2, D and E).
Therefore, this disappearance of glycogen in PCCA/ mice
appears to be due to a drastic consumption. Serum concentration of
either glucose or insulin was comparable in PCCA+/+ and
PCCA/ mice (data not shown), clearly indicating that
there was no defect in the glucose metabolism pathway that may cause a
complemental consumption of glycogen. In the kidney of
PCCA/ mice, the collecting ducts were enlarged with
marked incorporation of hyaline droplet casts, which implicated an
acceleration of reabsorption (Fig. 2, F and G).
All of these pathological changes in the liver and kidney of
PCCA/ mice might be primarily caused by the dehydration
and malnutrition due to no intake of milk after 10-12 h of age, since
similar phenotypes were also observed in PCCA+/+ mice that
had undergone dietary treatment for 16 h at the newborn period
(Fig. 2, H-K).
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Fig. 2.
Histological analysis of
PCCA / mouse. Liver sections from
PCCA+/+ (A) and PCCA/
(B) at 24 h of age were PAS-stained and analyzed.
PAS-positive products were identified as glycogen (see C,
red signals indicated by arrows) by a
glycogen-specific staining (14) of the PCCA+/+ sections.
Liver sections of both types of mouse embryos at E18.5 were also
PAS-stained and analyzed (D, E). PAS (+) products
are observed also in the PCCA/ embryo liver.
F, PAS-staining of the kidney sections of
PCCA+/+ and G, PCCA/. Enlarged
collecting ducts harboring the hyaline droplets in
PCCA/ kidney is indicated by arrows. PAS (+)
layer is also observed on the interior surface of the collecting duct
epithelium both in PCCA/ and PCCA+/+, which
is due to normal reabsorbing function by the collecting ducts.
H
K, wild-type (PCCA+/+) newborns had undergone
a dietary treatment for 16 h after birth (fed with
phosphate-buffered saline and kept in an incubator at 32 °C with
humidity), and then their liver and kidney were analyzed by
PAS-staining (H, J) together with samples from
mice that had been fed by maternal milk as a normal control
(I, K). After the dietary treatment the PAS (+)
products in the liver were almost disappeared (H), and the
collecting ducts in the kidney were enlarged and revealed incorporation
of hyaline droplets (J, indicated by arrows).
L, M, analysis for fat deposition (oil red O
staining) of the liver from PCCA+/+ or
PCCA/ at 24 h after birth. Red spots
indicate fat (lipid) droplets. Magnifications: ×400.
/
Mice--
Remarkable deposition of fat was observed in the liver of
PCCA/ mice at 24 h after birth (Fig. 2,
L and M). This fat-deposition was, however, not
detected at the late embryonic stage (E18.5) (data not shown), implying
that the deposition appeared to be rapidly generated in proportion to
the progression of acidosis after birth. Also in human, a few cases of
similar fat deposition in the liver of infant severe-form PA patients
have been also reported (17, 18). Accelerated dehydration and
malnutrition in addition to accumulation of propionyl-CoA might disturb
the general oxidation of fatty acids in the liver, perhaps resulting in
their accumulation and deposition in the liver tissue of
PCCA/ mice (17-23). There was, however, no difference
in RNA expression of various genes related to either synthesis or
oxidation of fatty acids, e.g. SREBP-1 (a transcription
factor), fatty acid synthase, glucose 6-phosphate dehydrogenas,
acyl-CoA oxidase, and carnitine palmitoyl CoA transferase in
PCCA+/+ and PCCA/ mice as assessed by
Northern blotting (data not shown). Further study will be required to
clarify the precise mechanism of the fat deposition in the liver. This
fat deposition in the liver does not seem to provoke hepatic
dysfunction because serum concentrations of various parameters for
hepatic damage, such as GOT, GPT, 
-GTP and bilirubin, were
within the normal range in PCCA/ mice and comparable
with those in PCCA+/+ mice (data not shown). Overall, the
PCCA/ mouse properly represents the severe form of PA
patients and thus is a useful animal model to test therapies for the disease.
/ Mice by a Weak, Liver-specific PCCA Expression
via Transgene--
By using PCCA/ mice, we attempted
to address the potential efficacy of PCC gene supplementation as an
alternative strategy that might prevent PA more efficiently. In
general, gene transfer via the DNA vaccination or the virus-based
vectors has been studied extensively (6-9, 24-26). It appears,
however, impossible to directly test either strategy in
PCCA/ mice because unlike human patients,
PCCA/ mice die very shortly (in 24-36 h) after birth,
and that is not sufficient time for transferred genes to yield
efficient expression. Thus, we designed a PCCA transgenic mouse system,
in which the transgene expression pattern is similar to that in
postnatal gene transfer therapies i.e. 1) the transgene will
not express during the embryonic stages, 2) the transgene will express
in a tissue specific fashion, and 3) the supplementation of PCC
activity per a tissue by the transgene will be within an achievable
level by gene therapies in general according to the previous reports of various other genes (27-30). To satisfy these conditions, we generated transgenic mice expressing PCCA under the regulation of the
serum amyloid P component (SAP) gene promoter based on the following characteristics of SAP promoter (11). First, the SAP promoter regulates
the target gene to express exclusively in the liver (11, 31, 32). The
liver appears ideal as a recipient organ for PCC gene transfer because
the liver tissue is one of the primary organs that metabolize the
odd-numbered amino acids and fatty acids. Second, it is known that a
transgene under the SAP promoter regulation starts its expression only
after birth, preserving a low expression level for the first few days
and increases its expression thereafter (31, 32). Hence, SAP-PCCA
transgenic mouse on a PCCA/ background
(SAP+PCCA/) appears to enable one to
address the therapeutic efficacy of supplementation of a low amount of
PCC activity in the liver during the newborn and the early infant periods.
/ background to obtain
SAP+PCCA/ mice. As expected, the transgene
expression was restricted in the liver in all of three transgenic mouse
lines as assessed by Northern blotting (data not shown). Two lines of
SAP+PCCA/ mice were analyzed together with
PCCA/ and PCCA+/+ littermate mice (both two
SAP+PCCA/ lines revealed identical
phenotypes). At birth, all of these types of mice were
indistinguishable and started sucking milk. After 24 h, most of
PCCA/ mice revealed severe ketonuria, and died as
described above. At the same time point, however,
SAP+PCCA/ mice were healthy and continued
sucking, thus their stomachs were full with milk like
PCCA+/+ mice. The ketouria of
SAP+PCCA/ mice was significantly milder
than that of PCCA/ mice (+ ~ ++ in
SAP+PCCA/ versus ++++ in
PCCA/). After 36 h, in contrast to
PCCA/ mice that had all died, all of
SAP+PCCA/ littermate mice had survived.
SAP+PCCA/ mice continued growing
comparatively in size with PCCA+/+ mice, and showed no
abnormalities in behavior, responses to various stimuli, sleep, and
sexuality. Both male and female adult
SAP+PCCA/ mice revealed no defect in
breeding efficiency.
/ mice at various ages. As
expected, no transgene expression, thus also no PCC activity, was
detected in the E12 embryonic liver. Transgene was not expressed even
at the late embryonic stage, E18.5, which is immediately before the
birth (data not shown). Twenty-four h after birth, the PCC activity in
the SAP+PCCA/ mouse liver was 10-20% of
that in the PCCA+/+ mouse liver, supplemented by a low
expression of the transgene (~20% of the level of the endogenous
PCCA mRNA expression in PCCA+/+ liver; compare with
24 h +/+ lane). The concentrations of both propionyl-carnitine and 3-HP in these mice were significantly decreased
as compared with those in PCCA/ mice. Thus,
supplementation of 10-20% PCC activity only in the liver was
sufficient to prevent the lethal fate of neonatal PCCA/
mice.
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Fig. 3.
PCC complementation in
SAP+PCCA / mice. PCCA mRNA
expression (transgene or endogenous) in the liver, PCC activity in the
liver, serum propionyl-carnitine and urine 3-HP concentrations, and the
level of urine ketone in SAP+PCCA/ mice at
various ages, and in some other types of mice (PCCA/
and PCCA+/+ mice) as controls, are presented. Northern
blotting was performed as described in the legend for Fig.
1B. / SAP+,
SAP+PCCA/ mouse; /,
PCCA/ mouse; +/+, PCCA+/+ mouse.
E12, embryonic day 12 (after fertilization); 24h, 24 h
after birth; 3w, 3 w of age. Each number is the average
of the results from 3-5 mice.
/ mice was preserved until 6-8
days of age, resulting in persistence of high propionyl-carnitine concentration and ketonuria, which were in the comparable levels to
those at 24 h after birth (data not shown).
SAP+PCCA/ mice, however, showed no growth
abnormality as compared with PCCA+/+ mice, as described
above. The transgene expression started to increase after 8-10 days of
age (data not shown), and at 3 weeks of age,
SAP+PCCA/ mice gained comparable PCC
activity in the liver with PCCA+/+ mice (Fig. 3). This
increase of PCC activity was followed by further decrease of
propionyl-carnitine and 3-HP concentrations. Thereafter, these
concentrations in SAP+PCCA/ mice were at
plateau levels similar to those at 3 weeks of age.
/ mice in 24 h after birth, was entirely
recovered by transgene expression, and was comparable in
SAP+PCCA/ and PCCA+/+ mice
either at 24 h or 3 weeks of age (Fig.
4, A-E). In addition, the
pathological change observed in the kidney of PCCA/
mice, the enlargement of collecting ducts with marked incorporation of
hyaline droplet casts, was decreased in
SAP+PCCA/ mice (Fig. 4, F-H).
These results may be explained mainly by the prevention of dehydration
and malnutrition in SAP+PCCA/ mice.
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Fig. 4.
Histological analysis of
SAP+PCCA / mice.
AH, PCCA+/+ (+/+), PCCA/
(/) as well as SAP+PCCA/ (/SAP+)
mouse were histologicaly analyzed by PAS-staining. Liver sections of
the three types of mice at 24 h after birth
(A--C) or of PCCA+/+ and
SAP+PCCA/ mouse at 3 weeks of age (D,
E). PAS (+) products are observed also in
SAP+PCCA/ mouse liver (C, E).
FH, kidney sections of the three types of mice at 24 h after birth. Enlargement of collecting ducts with an incorporation of
hyaline droplets observed in PCCA/ kidney
(G, indicated by arrows) is not detected in
SAP+PCCA/ kidney (H). PAS (+)
layer is also observed on the interior surface of the collecting duct
epithelium both in all types of mice, which is due to normal
reabsorbing function by the collecting ducts (also see the legend for
Fig. 2). Magnifications: ×400.
/ mice at 24 h of age, though
the severity was varied in each individual: some mice were entirely
free from the fat-deposition whereas others harbored the deposition
that was, however, obviously milder than that in PCCA/
mice (data not shown). Interestingly, at 3 weeks of age, most of
SAP+PCCA/ mice revealed no fat-deposition
in the liver (data not shown). These results clearly demonstrate that
the fatty-change of the liver is reversible, and in addition,
supplementation in the liver of at least 10-20% of the level of the
endogenous PCC activity is sufficient for decreasing the fat-deposition
in PCCA/ mice. The lack of marked dehydration and
malnutrition might have also contributed to the prevention of the fat
deposition in SAP+PCCA/ mice.
/ mouse revealed different
expression pattern of transgene, probably due to the influence of the
transgene integrating site in the chromosome. In this specific SAP+PCCA/ line
(SAPlow+PCCA/), unlike in the other two
lines of SAP+PCCA/ mice described above, a
low level of transgene expression, thus low PCC activity (10-20% of
that in PCCA+/+) in the liver, persisted even after 2 weeks
of age (data not shown). Although these
SAPlow+PCCA/ mice survived for the first 2 weeks, which is the newborn and early infant periods, they died before
3 weeks of age, demonstrating severe ketonutia (+++ ~ ++++) and
dehydration. This result strongly implicated that a larger amount of
PCC supplementation is required in proportion to the increase of
protein intake as mice grow. This result may also explain the diversity
of the age in the first onset of the severe ketoacidosis in human PA
patients, each of which possesses different PCC activity depending on
the type of PCC gene (either PCCA or PCCB)
mutation (2, 3, 33).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/, provided important information
as summarized bellow. First, a postnatal supplementation of at least
10-20% of endogenous PCC activity exclusively in the liver prevents
the fatal fit of severe ketoacidosis during the newborn and early
infant periods. Second, a persistent mild ketoacidosis (revealing ~6-
to 8-fold higher concentration of propionyl-carnitine than that in
wild-type mice and a mild ketonuria) appears not to essentially
influence infant development. This is reminiscent of the normal
embryonic development in PCCA/ mice, in which a certain
accumulation of propionic acid appeared to be present during the
embryonic period as described above. Lastly, 10-20% supplementation
of PCCA activity efficiently tempers the fat-deposition observed in the
liver of PCCA/ mice. Therefore, it is also very likely
that in human, the PCC gene transfer into the liver may be effective to
prevent the fatal acceleration of ketoacidosis in neonatal PA patients,
thus preserving normal growth during the early infant period. In
addition, the early death of SAPlow+PCCA/
mice, which harbored a persistent low level expression of
PCCA transgene even after the late infant stage, clearly
implicated that a larger amount of PCC supplementation appears to be
required in proportion to the growth of patients.
/
and SAPlow+PCCA/ mice, we propose a
two-step therapy to achieve efficient prevention of PA complaints in
human patients: a partial supplementation of PCC activity exclusively in the liver, followed by a high amount of PCC supplementation in the
late infant period. Because the "first step" treatment requires
partial (at least 10-20%) and transient (only during the newborn and
early infant periods) supplementation, a virus-based gene transfer in
the liver (of either PCCA or PCCB gene depending on which gene is defected in each patient) (34) may be a plausible strategy despite the following disadvantages implicated for the virus-based gene transfer. For example, 1) low supplementation efficiency (in a whole tissue) due to the low expression and limited distribution of the virus-infected area, and 2) difficulty of long term
persistence of expression due to the instability of the transfected
gene or production of antibodies against either the virus or the
introduced gene (35). For the "second step" therapy, a more drastic
treatment, such as liver-transplantation, which has been attempted so
far in two infant PA patients (5), might be required. The second step
therapy, however, will be performed in late infant patients who have
been well controlled by the first step therapy. This will lead to less
severe damage for patients than such a drastic therapy done in the
newborn or the early infant periods as the initial treatment. Judging
by the observation from SAP+PCCA/ mice,
whether the serum propionyl-carnitine concentration is within ~6- to
8-fold higher than that in the normal population, might be referred to
as a criterion to decide the frequency of the first step gene-transfer
treatment as well as the opportunity to perform the second step treatment.
/
mice showed no apparent clinical complaints without any dietary
limitation. This suggests that the two-step therapy in human PA
patients also might require no strict dietary limitation. It is,
however, strongly possible that more efficient therapeutic results may
be obtained if dietary limitation therapy is combined.
/ mice enabled us to observe the PA
progression in a kinetic fashion from various aspects, including
histology, which provided us new insights on the pathogenesis
contributing to the severe illness. In addition,
SAP+PCCA/ mice indicated the potential
efficacy of the two-step PCC supplementation therapy for human PA
patients to prevent the fatal acceleration of ketoacidosis, which
appears to result in essentially no clinical complaints even without a
strict dietary limitation. These two types of mice will be useful
animal models for further understanding of PA pathogenesis, as well as
to test a variety of strategies toward establishment of the thorough
therapy of PA.
| |
ACKNOWLEDGEMENTS |
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We are grateful to Drs. O. Sakamoto, and K. Iinuma (Tohoku Univ.), N. Hayashi, Y. Kato, and K. Yamauchi (Tokyo Women's Med. Univ.), and S. Ohnishi (Tokyo Univ.) for useful clinical suggestions and discussion; Drs. M. Iga (Shimane Univ.), H. Takeiri, and N. Sakayori (Tokyo Women's. Med. Univ.) for technical assistance; Drs. K. Araki and K. Yamamura (Kumamoto Univ.) for pSG-1 vector; and Dr. I. Shimomura (Univ. of Texas) for insulin and glucose analysis.
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FOOTNOTES |
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* 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: Center for Immunology, The Univ. of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., NA7200, Dallas, TX 75390-9093. Tel.: 214-648-7322; Fax: 214-648-7331; E-mail: Toru.Miyazaki@UTSouthwestern.edu.
Published, JBC Papers in Press, July 18, 2001, DOI 10.1074/jbc.M105467200
2 T. Miyazaki and T. Ohura, unpublished result.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 1. | Fenton, W. A., and Rosenberg, L. E. (1995) The Metabolic and Molecular Bases of Inherited Disease , pp. 1423-1440, McGraw-Hill, NY |
| 2. | Perez-Cerda, C., Merinero, B., Rodriguez-Pombo, Perez, B., Desviat, L. R., Muro, S., Richard, E., Garcia, M. J., Gangoiti, J., Ruiz-Sala, P., et al.. (2000) Eur. J. Hum. Genet. 8, 187-194 |
| 3. | Ugarte, M., Perez-Cerda, C., Rodriguez-Pombo, P., Desviat, L. R., Perez, B., Richard, E., Muro, S., Campeau, E., Ohura, T., and Gravel, R. A. (1999) Hum. Mutat. 14, 275-282 |
| 4. | Brandt, I. K., Hsia, Y. E., Clement, D. H., and Provence, S. A. (1974) Pediatrics 53, 391-395 |
| 5. | Saudubray, J. M., Touati, G., Delonlay, P., Jouvet, P., Schlenzig, J., Narcy, C., Laurent, J., Rabier, D., Kamoun, P., Jan, D., and Revillon, Y. (1999) Eur. J. Pediatr. 158, 65-69 |
| 6. | Wang, F., Raab, R. M., Washabaugh, M. W., and Buckland, B. C. (2000) Metab. Eng. 2, 126-139 |
| 7. | Tsai, S. Y., Schillinger, K., and Ye, X. (2000) Curr. Opin. Mol. Ther. 2, 515-523 |
| 8. | Morse, M. A. (2000) Curr. Opin. Mol. Ther. 2, 448-452 |
| 9. | Lamhonwah, A. M., Leclerc, D., Loyer, M., Clarizio, R., and Gravel, R. A. (1994) Genomics 19, 500-505 |
| 10. | Miyazaki, T., Hirokami, Y., Matsuhashi, N., Takatsuka, H., and Naito, M. (1999) J. Exp. Med. 189, 413-422 |
| 11. | Toyonaga, T., Hino, O., Sugai, S., Wakasugi, S., Abe, K., Shichiri, M., and Yamamura, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 614-618 |
| 12. | Gravel, R. A., Lam, K. F., Scully, K. I., and Hsia, Y. (1977) Am. J. Hum. Genet. 29, 378-388 |
| 13. | Novikoff, A. B., Novikoff, P. M., Rosen, O. M., and Rubin, C. S. (1980) J. Cell Biol. 87, 180-196 |
| 14. | Nowak, M. A., Fatteh, S. M., and Campbell, T. E. (1998) Arch. Pathol. Lab. Med. 122, 353-360 |
| 15. | Wolf, B., Hsia, Y. E., Sweetman, L., Gravel, R., Harris, D. J., and Nyhan, W. L. (1981) J. Pediatr. 99, 835-846 |
| 16. | Rosario, P., and Medina, J. M. (1982) J. Inherit. Metab. Dis. 5, 59-62 |
| 17. | Glasgow, A. M., and Chass, H. P. (1976) Pediatr. Res. 10, 683-686 |
| 18. | Linseisen, J., and Wolfam, G. (1993) J. Parenter. Enteral. Nutr. 17, 522-528. |
| 19. | Sperl, W., Murr, C., Skladal, D., Sass, J. O., Suormala, T., Baumgartner, R., and Wendel, U. (2000) Eur. J. Pediatr. 159, 54-58 |
| 20. | Wendel, U., Zass, R., and Leupold, D. (1993) Eur. J. Pediatr. 152, 1021-1023 |
| 21. | Hiltunen, J. K., Karki, T., Hassinen, I. E., and Osmundsen, H. (1986) J. Biol. Chem. 261, 16484-11693 |
| 22. | Arai, K., Kawaguchi, A., Saito, Y., Koike, N., Seyama, Y., Yamakawa, T., and Okuda, S. (1982) J. Biochem. (Tokyo) 91, 11-18 |
| 23. | Lyles, D. S., Randall, C. C., and White, H. B. (1977) Biochim. Biophys. Acta 487, 16-27 |
| 24. | Lundstrom, K. (2000) Intervirology 43, 247-257 |
| 25. | Pandha, H. S., Martin, L. A., Rigg, A. S., Ross, P., and Dalgleish, A. G. (2000) Curr. Opin. Mol. Ther. 2, 362-375 |
| 26. | Anklesaria, P. (2000) Curr. Opin. Mol. Ther. 2, 426-432 |
| 27. | Kozlowski, D. A., Bremer, E., Redmond, D. E., Jr., George, D., Larson, B., and Bohn, M. C. (2001) Mol. Ther. 3, 256-261 |
| 28. | Fan, X., Brun, A., and Karlsson, S. (2000) Gene Ther. 7, 2132-2138 |
| 29. | Wahlfors, J., Loimas, S., Pasanen, T., and Hakkarainen, T. (2001) Histochem. Cell Biol. 115, 59-65 |
| 30. | Smith-Arica, J. R., and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3, 43-49 |
| 31. | Zhao, X., Araki, K., Miyazaki, J., and Yamamura, K. (1992) J. Biochem. (Tokyo) 111, 736-738 |
| 32. | Takashima, H., Araki, K., Miyazaki, J., Yamamura, K., and Kimoto, M. (1992) Immunology 75, 398-405 |
| 33. | Richard, E., Desviat, L. R., Perez, B., Perez-Cerda, C., and Ugarte, M. (1999) Biochim. Biophys. Acta 1453, 351-358 |
| 34. | Connelly, S. (1999) Curr. Opin. Mol. Ther. 1, 565-572 |
| 35. | Hackett, N. R., Kaminsky, S. M., Sondhi, D., and Crystal, R. G. (2000) Curr. Opin. Mol. Ther. 2, 376-382 |
| 36. | Iga, M., Kimura, M., Ohura, T., Kikawa, Y. S., and Yamaguchi, S. (2000) J. Chromatogr. B Biomed. Appl. 746, 75-82 |
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