Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF PAX-2 FOR CONTROLLED
APOPTOSIS OR SURVIVAL OF CELLS
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to modulators of P~~-2
gene for controlled therapeutical apoptosis of cells or
therapeutical survival of cells.
(b) Description of Prior Art
PAX2 is a transcription factor critically
required during the development of the nervous and
excretory systems, including the mid/hindbrain, spinal
cord, eye, ear and urogenital tract (Dressler, G.R. et
al. (1990) Development, 109, 787-795; Eccles, M.R. et
al. (1992) Cell Growth & Diff. , 3, 279-289; Dahl, E. et
al. (1997) BioEssays, 19, 755-764). Like other members
of the PAX gene family, PAX2 encodes a conserved 128
amino acid paired box DNA binding domain in the N-
terminal portion of the molecule (Dahl, E. et al.
(1997) BioEssays, 19, 755-764). While PAX2 is a
transcriptional regulator, there are no proven target
genes regulated by PAX2, and little is yet known of the
exact role of PAX2 during development of the nervous or
excretory systems.
Eight of the nine Pax genes cause phenotypic
abnormalities when mutated in humans or mice, and in
four of these (including abnormalities caused by PAX2
mutations) developmental abnormalities are observed in
the heterozygotes, revealing haploinsufficiency (Dahl,
E. et al. (1997) BioEssays, 19, 755-764; Sanyanusin, P.
et al. (1995) Nature Genet., 9, 358-364). To date all
PAX2 mutations in humans have occurred within the
conserved paired box and octapeptide sequences
contained in the 5' half of the gene (refer to the
human PAX2 sequence variant database online)
(Sanyanusin, P. et al. (1995) Nature Genet., 9, 358-
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364). The 3' half of the PAX2 gene, which covers the
remaining 7 of the 12 exons including alternative
splices in exons 6, 10 and 12, encodes a putative
transactivation domain.
In previous reports, patients with PAX2
mutations have been noted to have optic nerve colobomas
and renal hypoplasia (renal-coloboma syndrome)
(Sanyanusin, P. et al. (1995) Nature Genet., 9, 358-
364). This syndrome has mostly been characterized
within the last 10 years, and is associated with a
number of less common features, including high
frequency hearing loss, seizure and brain malformation
disorders, joint and skin anomalies and vesico-ureteral
reflux (VUR) (Sanyanusin, P. et al. (1995) Nature
Genet., 9, 358-364). The renal phenotype associated
with renal-coloboma syndrome is frequently accompanied
by end-stage renal failure, often necessitating renal
transplant. While it is clear that PAX2 plays a
critical role during kidney development, the exact role
is unknown, and our present understanding of the
pathogenesis of renal failure as a result of PAX2
mutations is poor.
During embryonic life the ureteric bud emerges
from the nephric duct, growing outward into and
arborizing within the undifferentiated mesenchyme.
Signals from each branch of the ureteric bud induce
adjacent mesenchymal cells to transform into proximal
tubules and glomeruli of individual nephrons which
ultrafilter fluid from blood. Each nephron fuses with
its parent branch of the ureteric bud, providing an
outlet to the bladder. Thus the size and functional
capacity of each kidney is ultimately determined by the
complexity of ureteric bud branching and the number of
individual nephrons which have been induced by the time
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the pool of metanephric stem cells has been consumed
and nephrogenesis is terminated.
During normal kidney development PAX2 is
expressed throughout the branching ureteric bud,
Wolffian and Mullerian ducts. PAX2 is subsequently
expressed in each focus of induced nephrogenic
mesenchyme and its derivatives, the early epithelial
structures of the emerging nephron (Dressier, G.R. et
al. (1990) Development, 109, 787-795; Eccles, M.R. et
al. (1992) Cell Growth & Diff., 3, 279-289). The
importance of PAX2 to nephrogenesis is evident in mice
with targeted disruption or spontaneous mutations of
the Pax2 gene (Dahl, E. et al. (1997) BioEssays, 19,
755-764; Eccles, M.R. (1998) Pediatr. Nephrol., 12,
712-720). Heterozygous mutants have a phenotype very
similar to the human PAX2 mutation syndrome (renal-
coloboma syndrome) and are able to reproduce.
Homozygous Pax2 mutant mice lack kidneys, ureters, vas
deferens, epidydimis, seminal vesicles, uterus,
oviducts and vagina and have developmental defects of
the eyes, ears and CNS; these defects are lethal in the
perinatal period (Eccles, M.R. (1998) Pediatr.
Nephrol., 12, 712-720).
To gain insight into the cause of renal
abnormalities in patients with PAX2 mutations and to
understand how these abnormalities lead to renal
failure, we initially focused on the renal phenotype in
series of patients with renal-coloboma syndrome. A new
PAX2 mutation was identified in this study, which was a
novel stop codon mutation in PAX2 exon 7 in 9 members
of a large Brazilian pedigree spanning three
generations. We also identified the common 6619
insertion mutation in exon 2 of PAX2 in a sporadic
Japanese patient. The renal phenotypes in a total of 29
renal-coloboma syndrome patients were then compared,
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identifying renal hypoplasia as the most common
congenital renal abnormality. To characterize the
etiology of the renal hypoplasia, fetal kidneys of mice
lNeu
carrying a Pax2 mutation (Koseki, C. et al. (1992)
J. Cell Biol., 119, 1327-1333) were analyzed. This
mutation is identical to the 6619 insertion mutation in
some humans. The fetal kidney size of heterozygous
mutants was reduced to 600 of that of wild-type
littermates, closely resembling renal hypoplasia in
humans. Heterozygous lNeu mice showed reduced branching
and increased apoptotic cell death in fetal kidney
collecting ducts, but the increased apoptosis was not
associated with random stochastic inactivation of Pax2
expression in the mutant kidneys, and Pax2 was
biallelically expressed during kidney development. The
extent of the apoptosis correlated well temporally and
spatially with the known pattern of PAX2 expression,
tapering off when PAX2 expression levels are known to
decline. Our findings support the notion that
heterozygous PAX2 mutations lead to increased apoptosis
and reduced branching of the ureteric bud during a
critical window in kidney development.
It would be highly desirable to be provided with
modulators of PAX-2 gene for controlled therapeutical
apoptosis of cells or therapeutical survival of cells.
SZJNiMARY OF THE INVENTION
One aim of the present invention is to provide
modulators of PAX-2 gene for controlled therapeutical
apoptosis of cells or therapeutical survival of cells.
In accordance with the present invention there
is provided a method of treating cancer and cystic
kidney disease in a patient comprising the step of
administering to said patient a therapeutically
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effective amount of an agent which selectively prevents
the function of PAX-2.
The agent prevents the expression of PAX-2, such
as an antisense of PAX-2.
The agent may inhibit the activity of PAX-2,
such as a ribozyme.
Cancer treated is selected from the group
consisting of prostate, ovarian, bladder and kidney
cancers.
In accordance with the present invention there
is provided a method of rescuing cells from apotosis in
a patient comprising the step of administering to said
patient a therapeutically effective amount of an agent
which selectively stimulate the function of PAX-2.
The agent may activate the expression of PAX-2.
In accordance with the present invention there
is provided a method to enhance resistance of normal
tissues to apoptotic cell death induced by chemotherapy
or radiation therapy in a patient comprising the step
of administering to said patient a therapeutically
effective amount of an agent which selectively
stimulate the function of PAX-2.
The agent may activate the expression of PAX-2.
In accordance with the present invention there
is provided a genetic construct comprising a nucleic
acid encoding a molecule capable of preventing or
stimulating the function of PAX-2.
In accordance with the present invention there
is provided a method of diagnosing prostate, ovarian, ~'
bladder and/or kidney cancer, renal hypoplasia, renal
failure, renal-coloboma and cystic kidney diseases in a
patient comprising the steps of
a) obtaining a sample containing nucleic acid or
protein from prostate, ovary, bladder and/or
kidney cells or tissue of said patient; and
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b) determining whether the sample contains a PAX-2
mutation or a level of PAX-2 nucleic acid or
protein asscciated with inappropriate postnatal
PAX-2 overexpression, thereby being indicative
of neoplastic phenotype or disease phenotype
associated with abnormal PAX-2 function.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the pedigree of the Brazilian
family with renal-coloboma syndrome;
Fig. 2 illustrates the detection of PAX2
mutations in the Brazilian family, and in a sporadic
Japanese patient;
Fig. 3 illustrates a graph of kidney size in
10
patients with PAX2 mutations;
Fig. 4 illustrates the morphology of E15 lNeu
mutant and wild type kidneys;
Fig. 5 illustrates the analysis of nephrogenesis
in E15 lNeu mutant and wild type kidneys;
Fig. 6. illustrates the analysis of apoptosis
lNeu
and cell proliferation in fetal kidneys from Pax2 /+
heterozygous mutant and wild type mice;
Fig. 7 illustrates graphs showing the number
of
nick-end-labeled cells per kidney area in fetal
and
postnatal kidneys from Pax2lNeu/+ mutant and wildtype
mice; and
Fig. 8 illustrates biallelic expression of Pax2
in fetal kidney epithelial cells;
DETAILED DESCRIPTION OF THE INVENTION
PAX2 mutations cause renal-coloboma syndrome
(RCS), a rare multi-system developmental abnormality
involving optic nerve colobomas and renal
abnormalities. End-stage renal failure is common in
RCS, but the mechanism of how PAX2 mutations lead to
renal failure is unknown. PAX2 is a member of a family
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of developmental genes containing a highly conserved
"paired box" DNA-binding domain, and encodes a
transcription factor expressed primarily during fetal
development in the CNS, eye, ear and urogenital tract.
Presently, the role of PAX2 during kidney development
is poorly understood. To gain insight into the cause of
renal abnormalities in patients with PAX2 mutations,
kidney anomalies were analyzed in patients with RCS,
including a large Brazilian kindred in whom a new PAX2
mutation was identified. In a total of 29 patients
renal hypoplasia was the most common congenital renal
abnormality. To determine the direct effects of PAX2
mutations on kidney development fetal kidneys of mice
lNeu
carrying a Pax2 mutation were examined. At E15,
heterozygous mutant kidneys were about 600 of the size
of wild-type littermates, and the number of nephrons
was strikingly reduced. Heterozygous lNeu mice showed
increased apoptotic cell death during fetal kidney
development, but the increased apoptosis was not
associated with random stochastic inactivation of Pax2
expression in mutant kidneys; Pax2 was shown to be
biallelically expressed during kidney development.
These findings support the notion that heterozygous
mutations of PAX2 are associated with increased
apoptosis and reduced branching of the ureteric bud,
due to reduced PAX2 dosage during a critical window in
kidney development.
Materials and Methods
PCR-SSCP and detection of PAX2 mutations
Genomic DNA from each individual was extracted
from peripheral blood (collected with informed consent)
using a DNA extraction kit (Promega). Fragments
spanning exons 1-12 of PAX2 were amplified from genomic
DNA using PCR primers in the introns flanking the
exons. The PCR products were labeled by incorporation
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of 32P-dCTP in reactions containing 100 ng DNA, 62.5 ~M
dNTPs, 1 ~Ci [a-32P] dCTP (3000 Ci/mmol), 20 pmol of
each primer, reaction buffer (50mM KCl, lOmM Tris
pH 8.3, 1.0-3.0 mM MgCl2) and Pwo (Boehringer) or
AmpliTaq Gold (Perkin Elmer) DNA polymerase, and
electrophoresed in non-denaturing 60 or 120
polyacrylamide gels as described previously
(Sanyanusin, P. et al. (1995) Nature Genet., 9, 358-
364) to reveal single-strand conformational variants.
Subcloning and DNA sequencing
PCR products were directly sequenced using y33p_
ATP radiolabeled kinased primers and a cycle sequencing
kit (GIBCO-BRL). For subcloning of exon 2 and exon 7
PCR products, mutant and normal alleles of PAX2 were
amplified using Pwo DNA polymerase (Boehringer
Manheim), and subcloned into EcoRV digested pBluescript
II (Stratagene). Positive clones were identified by
blue/white selection, and plasmid DNA was isolated
using Wizard mini-preps kits (Promega). Sequencing
reactions were carried out on plasmid DNA as described
previously (Sanyanusin, P. et al. (1995) Nature Genet.,
9, 358-364).
Mouse breeding, genotyping and embryo preparation
Wild type and lNeu mouse colonies were bred and
maintained at Neuherberg, Germany and at University of
Otago, New Zealand. Timed matings of male mice
heterozygous for the Pax2lrreu mutant allele (Koseki, C.
et al. (1992) J. Cell Biol., 119, 1327-1333) were
carried out with female C57BL/6 mice, and the embryos
were genotyped. Genotyping was performed as described
(Koseki, C. et al. (1992) J. Cell Biol., 119, 1327-
1333), or by PCR amplification of genomic DNA extracted
from tail slices by using primers 2F and 2R (see
Fig. 8). PCR amplification reactions were as
previously described (Sanyanusin, P. et al. (1995)
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Nature Genet., 9, 358-364). The presence of a 167 by
fragment in addition to a 166 by fragment when the PCR
products were electrophoresed on a 6o polyacrylamide
gel indicated the presence of the mutant Pax2 allele.
Fetal kidneys (E15-E18) were dissected from
heterozygous Pax2lNeu/+ mutant or wild-type mice. The
expression of the mutant Pax2lNeu allele was
demonstrated in fetal kidney RNA from Pax2lNeu/+ mutant
mice by RT-PCR amplification of total kidney RNA using
primers 1F and 3R, spanning exons 1-3 of Pax2. Nested
PCR amplification of the RT-PCR product from exons 1-3
was carried out using primers 2F and 2R. PCR products
were electrophoresed on 6o polycrylamide gels. Primer
sequences were based on the murine Pax2 sequence
(Dressier, G.R. et al. (1990) Development, 109, 787-
795) and were as follows;
1F, 5'-CCA CCG TCC CTC CCT TTT CTC CT-3'~
2F, 5' GGG CAC GGG GGT GTG AAC CAG-3';
2R, 5'-CTG CCC AGG ATT TTG CTG ACA CAG CC-3'~
3R, 5'-CTG TGT CAT TGT CAC AGA TGC CCT CG-3'.
Microscopic analysis
Heterozygous PAX2 mutant E15 embryos and their
wild type littermates were incubated in freshly
prepared 4o paraformaldehyde in PBS for 16-18 hours and
stored at 4° in 70o ethanol prior to embedding in
paraffin. Serial 5 ~m sagital sections of both kidneys
from each mutant and wild type embryo were prepared on
Superfrost/Plus slides (Fisher). Sections were stained
with hematoxylin-eosin and analyzed by bright field
microscopy: the number of glomeruli and early
epithelial structures (renal condensates, comma- and S-
shaped bodies) in the nephrogenic layer was counted in
serial cross-sections from both kidneys of lNeu and
wild type embryos. To compare the maximal cross-
sectional area of heterozygous and wild type kidneys,
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serial cross-sections were visualized by
photomicroscopy and projected on a television screen.
The outline of each renal image was traced onto paper,
cut out and weighed to estimate relative surface area.
The maximal cross-sectional area for each kidney was
plotted in arbitrary units for inter-kidney
comparisons.
PAX2 immunostaining
Polyclonal anti-mouse PAX2 antibody was
purchased from Zymed, California. Mouse sections were
deparaffinized and incubated with primary antibody
(10 ug/ml) for 30 min. at room temperature in PBS
supplemented with 20mM glycine and to BSA. PAX2
antibody was detected with Vecstatin ABC Rabbit IgG kit
(Vector Laboratories) as described by the manufacturer,
followed by incubation with DAB substrate (Sigma).
Sections were washed in PBS, counterstained with
hematoxylin, dehydrated and mounted with Permount
(Fisher). V~lild type and mutant tissues were stained
simultaneously; each analysis was repeated 3 times.
Detection of apoptosis (TUNEh staining)
The TUNEL assay was performed on paraffin-
embedded tissue sections as described (Gavrielli, Y. et
al. (1992) J. Cell Biol., 119, 493-501) with
modifications. The paraffin sections were dewaxed,
rehydrated, partially digested with proteinase K
(15 ~g/ml; Boehringer Mannheim) in Tris~C1 (10 mM, pH
7.6) at room temperature (RT) for 15 minutes. After the
endogenous peroxidase was inactivated with 2o H202 for
10 minutes at RT, the sections were incubated for 90
minutes at 37°C with terminal deoxynucleotidyl
transferase (TdT, Boehringer Mannheim) in TdT reaction
buffer (1mM dCTP, 1mM dGTP, 1mM TTP, 1mM biotin-14-
dATP, 140 mM sodium cacodylate, 1 mM cobalt chloride,
30 mM Tris-HC1, pH 6.4). The sections were then washed
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3 times in PBS, incubated with extravidin peroxidase
(1:250 dilution in PBS, SIGMA) for 60 minutes at RT,
rinsed 3 times in PBS, stained with diaminobenzidine
(Boehringer Mannheim) for 5 minutes at RT, and
counterstained with to methyl green.
BrdU incorporation studies and PCNA immunostaining
BrdU incorporation studies were performed
exactly as described (Gavrielli, Y. et al. (1992) J.
Cell Biol., 119, 493-501). For PCNA immunostaining
mouse sections were deparaffinized, hydrated, and
endogenous peroxidase activity was quenched by
incubating in methanol containing 0.3o H202. Slides
were incubated in 2.5 ~g/ml PCNA monoclonal antibody
(Oncogene Research) at room temperature for 30 min
followed by two brief washes in PBS. PCNA antibody was
detected with Vecstatin Mouse IgG kit (Vector
Laboratories) as described by the manufacturer followed
by incubation with DAB substrate (Sigma).
Results
Renal abnormalities in 29 patients with PAX2 mutations
To further characterize the renal phenotype
associated with renal-coloboma syndrome, renal features
were compared in a total of 29 patients with PAX2
mutations, including 10 new patients from 2 families.
The 10 new patients are described in the following. A
three generation Brazilian kindred (pedigree shown in
Fig. 1) was evaluated for optic nerve and kidneys
defects consistent with diagnosis of renal-coloboma
syndrome. Individuals in three generations of the
pedigree (I, II and III) are shown in Fig. 1. Filled
symbols represent affected individuals. Open symbols
represent unaffected individuals.
The associated phenotypic abnormalities are
listed in Table 1. PCR-SSCP revealed a variant pattern
in exon 7 that was present in 5 of the affected members
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of the family, but was not present in 18 unaffected
members (Fig. 2A). PCR-SSCP analysis of PAX2 exon 7
showed the SSCP variant band in the affected
individuals, but not in the unaffected individual
(arrow; Fig. 2A).
Independently derived mutant alleles from the
exon 7 PCR product contained a cytosine to thymine
substitution at position 1289 (Fig. 2B). The mutant and
normal PAX2 sequence were identified in exon 7 of
affected members of the Brazilian family. The mutation
(arrowhead) is a C -> T substitution at position 1289
(underlined) of the PAX2 cDNA sequence (Eccles, M.R. et
al. (1992) Cell Growth & Diff., 3, 279-289) (Fig. 2B).
Normal alleles were also identified in DNA from
these individuals, while unaffected patients did not
contain the mutation. In exon 7 the nucleotide
substitution at this position resulted in the
disruption of a CacBI restriction endonuclease site due
to the change from a GCNNGC recognition motif to
GTNNGC. Restriction digestion with Cac8I of exon 7 PCR
products from one of the affected Brazilian family
members showed sequences resistant to digestion as well
as sequences which could be digested by Cac8l. Fig. 2C
shows the Cac81 restriction enzyme digestion of the PCR
products from affected and unaffected members of the
Brazilian family. The undigested band in the uncut
unaffected, and affected lanes and in the Cac81
digested affected lanes is 234 bp, which is digested to
products of 137 and 97 by by Cac8l.
Unaffected family members contained only
sequences that digested with Cac8l. Additionally, a
Japanese patient was examined who had sporadic
occurrence of optic nerve colobomas, renal anomalies
and bilateral cryptorchidism (see Table 1, patient
X2003 for phenotypic details).
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Table 1
Summary of phenotypic abnormalities in 9 members of the Brazilian
family
Patient Age Sex Phenotype
I-1 70 M Abdominal US normal, systemic hypertension, serum creatinine
1.8 mg/dL, blood urea 116 mg/dL, left mild sensorineural hearing
loss, optic pit right eye, optic nerve coloboma left eye, left eye
visual acuity 20/20, right eye counting fingers (CF).
II-2 deceased F History of a probable renal disorder
II-5 37 M Abdominal US showed bilateral small kidneys, and cortical
hyperechogenicity, renal failure over past 2 years, bilateral optic
nerve colobomas, visual acuity was CF in OD and 20/20 in OS.
II-7 40 F No renal dysfunction, abdominal US normal, bilateral optic nerve
colobomas, visual acuity was 20/20 in both eyes.
II-9 39 M History of bilateral nephrolithiasis, previous episode of
obstructive
urolithiasis in left ureter, single functioning right kidney, serum
creatinine 1.7 mg/dL, blood urea 54 mg/dL, bilateral optic nerve
colobomas, visual acuity was 20/20 in OD and CF in OS.
III-9 14 M No renal dysfunction, abdominal US normal, bilateral optic
papillae dysplasia, visual acuity 20/20 in both eyes.
III-11 18 M No renal dysfunction, abdominal US normal, bilateral optic nerve
colobomas, visual acuity 20/20 in both eyes.
III-13 21 F Abdominal US showed bilateral small kidneys with cystic
appearance in upper pole, hyperechogenicity of renal cortex and
no medullary differentiation, plasma creatine 8.5 mg/dL, blood
urea 142 mg/dL, bilateral optic nerve colobomas, and hypoplastic
optic nerve in left eye. She had also a rarefied retina. Visual acuity
was 20/200 in OD and CF in OS, history of febrile seizures with
cognitive impairment in childhood.
III-16 4 M Vesicoureteral reflux at birth, bilateral optic nerve pits, visual
acuity 20/20 in both eyes.
III-17 6 M Abdominal US showed bilateral small kidneys, bilateral cortical
hyperechogenicity, poor corticomedullary differentiation, was
submitted to renal transplantation in the beginning of 1998 (age 7)
and is doing very well, serum creatinine 5.6 mg/dL, blood urea
124 mg/dL, bilateral optic nerve colobomas, visual acuity was
20/60 OD and 20/200 OS.
x2003 13 M Small kidneys with multiple small cysts on CT and MRI,
progressive renal failure, bilateral cryptorchidism, bilateral optic
nerve coloboma with retinal detachment.
As above, each exon of the PAX2 gene was
amplified by PCR to identify a PAX2 mutation. A
variant SSCP pattern was observed for exon 2, and
sequencing of this PCR product revealed a guanosine
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nucleotide insertion. at position 619 (Fig. 2D). The
mutant and normal PAX2 sequence in exon 2 of the
Japanese patient are shown in Fig. 2D. The mutation
(arrowhead) is a G insertion resulting in an additional
G (underlined) in a homonucleotide tract of 7 G's.
A list of the renal abnormalities identified in
all 29 patients with PAX2 mutations, including the
patients in this study, is given in Table 2.
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Table
2
Renal
abnormalities
in
29
patients
with
PAX2
mutations
Patientreferenceage small solitaryrenal failure, VUR renal
kidneys)kidney proteinuria, transplant
or abnormal
histology
1 5 15 + - + + +
2 5 10 + - + + -
3 5 6 + - + + +
4 5 35 - - + - -
430 7 5 - + + - +
431 7 12 - - + - -
TRN 8 5 - - + - -
579 9 11 + - + - -
656 9 48 + - + - +
657 9 25 + - + - +
2646 9 20 + - + - +
III-8 10 70 - - + + -
IV-2 11 52 + - + - +
IV-3 11 46 + - + _ +
IV-6 11 40 + - + _ +
IV-7 11 35 + - + - -
V-2 11 16 + - + - -
F2 11 17 + - + + -
985 11 6 - - + + -
I-1 this 70 - - + - -
study
II-5 this 37 + - + - -
study
II-7 this 40 - - - - -
study
II-9 this 39 - - + - -
study
III-9 this 14 - - - - -
study
III-11 this 18 - - - - -
study
III-13 this 21 + - + - -
study
III-16 this 4 - - + + -
study
III-17 this 6 + - + - +
study
X2003 this 13 + - + - -
study
SUBSTITUTE SHEET (RULE 26)
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The most common renal abnormalities in the 29
patients were renal failure with histological
abnormalities, proteinuria, and renal hypoplasia (small
kidneys). Renal failure, histological abnormalities and
proteinuria are features commonly seen in association
with other disease processes, and are not specific for
renal-coloboma syndrome. These abnormalities were
probably secondarily acquired since birth. On the other
hand, renal hypoplasia, although seen in association
with a number of congenital conditions, is caused by
failure of embryonic growth of the kidney, and would
therefore be consistent with an abnormality arising
primarily from the PAX2 mutation. Small kidneys were
found in 17/29 patients (590), and in 10 of these
patients the sizes of the kidneys were measured by
ultrasound and graphed as a percentage of the mean
normal size of kidneys for individuals at that age
(Fig. 3). In Fig. 3 kidney lengths are shown as a
percentage of the mean normal kidney length for a
person of the same age and sex; black bar, left kidney;
grey bar, right kidney. The patients in order were 1,
3, TRN, 579, III-8, IV-2, IV-3, IV-6, IV-7, V-2 in
Table 2.
Renal hypoplasia was observed even in the
youngest patients in this study, consistent with it
being a congenital abnormality associated with renal-
coloboma syndrome.
Detailed analysis of the cause of the renal
hypoplasia associated with renal-coloboma syndrome
would have required characterization of fetal kidneys
from these patients. This was not possible in human
subjects, but a suitable mouse model of renal-coloboma
lNeu
syndrome (Pax2 ) was available in which the mutation
and syndrome were identical to those described in some
humans (Koseki, C. et al. (1992) J. Cell Biol., 119,
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1327-1333). We therefore undertook a detailed study of
lNeu
the kidney phenotype in heterozygous Pax2 mutant
fetal mice.
lNeu
Renal hypoplasia in Pax2 heterozygous mouse embryos
lNeu
Homozygous Pax2 mice have renal agenesis,
bilateral optic nerve colobomas, abnormalities of ear
development and missing midbrain-hindbrain regions.
These animals die within 24 hours of birth. However,
heterozygous lNeu mice, like their human counterparts,
are viable and have no gross midbrain-hindbrain
phenotype. Their main characteristics are abnormalities
of optic nerve development and hypoplastic kidneys (u).
Fetal mice carrying a heterozygous Pax2 mutation were
lNeu
obtained by crossing heterozygous Pax2 males with
C57BL/6 females, and the fetuses were genotyped.
Microscopic analysis of hematoxylin-eosin- stained
sections from each embryo of three litters revealed
that approximately 600 of the animals had hypoplastic
kidneys with maximal cross-sectional surface area
ranging from 30-750 of that of wild types.
Representative litter is presented in Figs. 4 and 5.
Representative maximal cross-sections of E15 embryos
from lNeu +/- mutant and wild type littermates were
stained with hematoxylin-eosin. Wild type kidneys show
a complex nephrogenic zone and maturing medullary core
(Fig. 4A). In contrast, lNeu kidneys are clearly
smaller with fewer nephrons and primitive medulla (Fig.
4B). Note the reduced number of mesenchymal condensates
and ureteric bud branches in the nephrogenic zone of
mutant kidney; mature glomeruli are absent on this
representative section. Magnification X 50. Maximal
cross-sectional area of wild type and lNeu heterozygous
mutant kidneys were measured in a representative litter
(arbitrary units; Fig. 5A). Mutant kidneys are smaller
(600 of the average wild type cross-sectional area) but
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overlap with the smallest wild type kidneys. Numbers of
mature glomeruli and early epithelial structures were
measured in maximal cross-sections of kidneys from a
representative litter Fig. 5B). Mutant kidneys have
fewer mature glomeruli (220 of wild type) and early
nephron structures (470 of wild type).
The largest heterozygous mutant kidneys
overlapped in size with the smallest kidneys from wild
type littermates. Unilateral renal agenesis was
encountered in approximately to and cystic
abnormalities were additionally observed in
approximately to of animals.
The nephrogenic zones of fetal (E15-E16) lNeu
kidneys were thin and contained fewer nephrons in
comparison to those of normal littermates. The number
of early epithelial structures derived from induced
metanephric mesenchyme (vesicles, comma- and S-shaped
bodies) was reduced to 30-400 of normal (Fig. 5b). The
number of mature glomeruli was even more sharply
reduced (20% of the wild type). Interestingly, early
tubular structures and glomeruli in Pax2 mutant kidneys
appeared to be of normal size and morphology (Fig. 4).
lNeu
Renal hypoplasia in Pax2 mace is associated with
enhanced apoptosis of the ureteric epithelium
To gain insight into the mechanism of renal
hypoplasia in Pax2 mutant mice, we examined the
patterns of apoptotic cell death and cell proliferation
in mutant and wild type fetal kidneys. TUNEL staining
was used to investigate whether the Pax2 mutation in
lNeu
heterozygous Pax2 mutant mice was associated with
increased apoptosis. An increase in TUNEL-positive
staining was observed in the kidneys of embryonic day
lNeu
15-16 (E15-16) Pax2 /+ mutant mice compared with in
homozygous wild-type littermates (Fig. 6A, 6B).
Demonstration of apoptosis (arrowheads) in the
collecting duct epithelia of kidneys from embryonic day
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15 (E15) Pax2lNeu/+ mutant mice (Fig. 6A), as compared
with wild type E15 mice (arrows) (Fig. 6B) by TUNEL
(TdT-mediated dUTP nick end labeling) staining
(magnification 100X). Analysis of cell proliferation
using BrdU incorporation (arrows) in the cortical
lNeu
region of kidneys from E15 Pax2 /+ mutant (Fig. 6C)
and E15 wild type mice (Fig. 6D). The overall level of
BrdU-labeling in mutant kidneys was similar to that in
wild type kidneys (magnification 250X).
The rate of cell proliferation, as analyzed by
BrdU uptake into DNA in the developing kidneys of
lNeu
mutant Pax2 /+, was equivalent to that of wildtype
mice from embryonic day E15 through postnatal day 6
(Fig. 6C, 6D). Similarly, PCNA staining was unchanged
in E15 lNeu embryos compared with their wild type
littermates. These results suggest that heterozygosity
for the lNeu mutation is associated with an increase in
the rate of apoptotic cell death but not with a
difference in the pattern of fetal kidney cell
proliferation.
Apoptotic cell death in the mutant kidneys was
examined in greater detail to identify the stages of
development in which apoptosis was increased and the
cell types involved. Although there were consistently
more TUNEL-positive cells (p<0.001) in the total
lNeu
kidneys of E15-E16 Pax2 /+ mutant kidneys than in
homozygous wild type littermates, this difference was
less noticeable at later stages, and was confined to
the collecting ducts (see below). In total kidneys of
day E18, newborns (1 day-old) , and in 5-6 day-old pups
the number of TUNEL-positive cells were not
significantly different in mutant and wild-type kidneys
(Fig. 7A). The graphs show the number of TUNEL-stained
lNeu
cells in Pax2 /+ mutant (white bars) and wildtype
mice (black bars) for (Fig. 7A) over the whole kidney,
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(Fig. 7B) specifically in collecting ducts, or
(Fig. 7C) specifically in renal cortex. The X-axis
shows mice analyzed at 3 different timepoints; E15-E16
(n=13 mutant and 9 wild type kidneys); E18-1 day old
(d.o.) (n=13 mutant and 9 wild type kidneys); and 5-6
d.o (n=9 mutant and 11 wild type kidneys).
By counting apoptotic cells in specific renal
lNeu
structures of Pax2 /+ mutant kidneys a significant
increase (p=0.0013) in TUNEL-positive staining was
identified in the collecting ducts and renal pelvis in
lNeu
fetal (E15 - E16) Pax2 mutants (Fig. 7b). This
difference was still detectable in E18 , but not in 5-6
day-old mice. TUNEL staining was also increased in the
lNeu
renal cortex in E15-E16 Pax2 /+ mutant fetal kidneys
(Fig. 7C), which mostly corresponded to ureteric buds.
Increased apoptosis was not observed in glomeruli, or
proximal or distal tubules at either E15-E16 or later
in development. The rate of apoptosis in fetal lNeu
kidneys was not determined prior to E15 because
successfully mated females did not have vaginal plugs,
and excessive numbers of randomly pregnant female mice
would have been required to obtain the Figures for
apoptotic rates.
lNeu
Pax2 mutant kidneys contain transcripts of both PAX2
alleles and express a reduced level of PAX2 protein
Recent evidence by others suggests that another
PAX gene, PAXS, is normally expressed stochastically
from only one allele in developing lymphocytes. This
observation raises the question as to whether PAX2
expression in developing kidney is also monoallelic.
Accordingly, heterozygous mutant kidney would be
comprised of a mixture of normal and null mutant cells.
Apoptotic cell death might occur in those cells
expressing the mutant allele, and viable collecting
duct cells could consist of those randomly expressing
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normal amounts of PAX2 protein from the wild type
allele. Alternatively, if PAX2 is expressed from both
lNeu
alleles, then nephrons of heterozygous Pax2 kidneys
should express PAX2 protein with perhaps a slight
reduction in intensity.
To test the hypothesis that renal hypoplasia
and the increased rate of apoptosis in lNeu mutant
fetal kidneys was associated with monoallelic Pax2
transcription we analyzed allelic patterns of Pax2
expression. The insertion mutation was used to
discriminate the mutant and wild type alleles (Fig. 8A)
in mRNA from heterozygous lNeu mutant mouse kidneys
following RT-PCR. Sequences depicting the G-insertion
lNeu
mutation in exon 2 of the Pax2 allele, the wild type
Pax2 allele, and the locations of the primers (1F, 2F,
2R, 3R) used for PCR and RT-PCR. Reverse transcribed
RNA was amplified using primers 1F and 3R (DNA
amplification was precluded by introns 1 and 2).
Primers 2F and 2R were used to amplify genomic DNA.
We reasoned that if Pax2 transcription were
monoallelic in the developing kidney, cells randomly
expressing the mutant allele might have a selective
disadvantage and represent a diminishing portion of the
kidney as development progressed. However, we found
that levels of mutant and wild type Pax2 mRNA (alleles)
were equivalent at all stages of development between
fetal (E 12) and postnatal (day +1) kidney (Fig. 8B),
suggesting that there was no selection for the favored
expression of one allele in mutant kidneys. 60
denaturing PAGE gel showing mutant and wildtype Pax2
alleles in DNA and RNA (arrows). Lanes 1, 2,
heterozygous lNeu genomic DNA (lane 1) and wild type
genomic DNA (lane 2); Lanes 3-9, RT-PCR of wild type
(lane 3) and heterozygous mutant lNeu fetal kidney mRNA
(lanes 4-9). E12 fetal kidneys were in lanes 3, 4; E14
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fetal kidneys were in lanes 8, 9; E18 fetal kidneys
were in lanes 6, 7; postnatal (day +1) kidney was in
lane 5.
In contrast to the above reasoning, cells
monoallelically transcribing the mutant Pax2 allele
might successfully proliferate, but these cells would
not express the normal PAX2 protein. If there were
large numbers of cells in the ureteric buds in the
mutant kidneys not expressing immuno-reactive Pax2
protein, then this would be consistent with the notion
that they express Pax2 mono-allelically from the mutant
allele. We would be able to detect the resultant
mosaicism (i.e. cells staining positively and
negatively for PAX2 protein) by performing
immunohistochemistry with a polyclonal anti-murine Pax2
antibody that reacts with C-terminal epitopes present
in wild-type Pax2 protein, but which are absent in the
truncated mutant Pax2 protein. Immunohistochemical
staining for Pax2 protein was observed in all
identifiable nuclei of ureteric bud and collecting duct
cells of heterozygous lNeu fetal kidneys (Fig. 8C), but
the intensity of Pax2 staining was uniformly less than
that in fetal kidneys of wild-type littermates analyzed
simultaneously. Immunohistochemistry of E15
heterozygous mutant lNeu mouse fetal kidney, and Fig.
8C D, E15 wild type mouse fetal kidney using anti-Pax2
primary antibody. Nuclei stained brown in the sections
correspond to cells of the collecting duct and ureteric
bud expressing the Pax2 protein.
By comparison, strong staining for Pax2 was
observed in all portions of wild type ureteric buds and
collecting ducts (Fig. 8D). This pattern of staining
was observed in each of three wild-type and three
mutant embryos analyzed simultaneously. These results
suggest that all the cells of the ureteric buds
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expressed the wild-type Pax2 allele. To examine PAX2
allelic expression patterns directly in clonal cell
populations we analyzed transcription in Wilms tumors
and in cloned renal carcinoma cells using a
polymorphism in human PAX2 (Fig. 8E). Depiction of the
C/A polymorphism in exon 8 used to distinguish PAX2
alleles during amplification of human PAX2 exon 8 mRNA
from Wilms tumors and renal carcinoma cells, and the
locations of the primers (P1, P2, P3, P4) used for RT-
PCR and DNA PCR. Reverse transcribed RNA was amplified
using primers P1 and P4 (DNA amplification was
precluded by introns 7 and 8), and RT-PCR products were
analyzed by SSCP. Genomic DNA for genotyping was
amplified using primers P2 and P3.
RT-PCR-SSCP was used to discriminate between
the two PAX2 alleles (Fig. 8f, lanes 1-3). Biallelic
transcription of PAX2 in fetal kidney (Fig. 8F, lane 7)
was not unexpected since monoallelic transcription of
PAX2 in one cell (if present) would be statistically
matched by monoallelic transcription of the opposite
allele in another cell. RT-PCR-SSCP gels showing
biallelic PAX2 expression in human fetal kidney and
tumors. SSCP revealed four conformations for the two
alleles (upper and lower arrows). Lanes 1-3, fetal
kidneys homozygous for one allele (lane 1),
heterozygous (lane 2), or homozygous for the other
allele (lane 3). Lanes 4-6, heterozygous Wilms tumors.
Lane 7, heterozygous human fetal kidney (12 weeks
gestation). Lanes 8, 9, renal carcinoma cell line
(A704) before cell cloning (lane 8), and after cell
cloning (lane 9). Primer sequences; exon 8; P1, 5'-AGC
TTT GGA TCG GGT CTT TGA-3'; P2, 5'-CCT TTC TCT GTG CGT
GCA TCA ATA GA-3'; P3, 5'-GGC ACC CTC CAC TGA ACG CAG-
3'; P4, 5'-CAG GGT GGA GGT GGG GTA G-3'.
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However, Wilms tumors are predominantly
comprised of clonal blastemal and epithelial tissues,
derived originally from embryonic kidney. Biallelic
PAX2 transcription was observed in mRNA isolated from a
series of 3 Wilms tumor tissues (Fig. 8f, lanes 4-6).
In addition, biallelic (albeit slightly unbalanced)
PAX2 transcription was observed in clonally expanded
renal carcinoma cells (Fig. 8F, lanes 8, 9) . The small
imbalance of PAX2 allelic expression observed in the
cells may be due to alterations in the number of copies
of each PAX2 allele in the cells, and was an indication
that the RT-PCR-SSCP technique was sensitive enough for
this purpose.
Discussion
In accordance with the present invention, we
have further characterized the renal phenotype
associated with renal-coloboma syndrome and have
identified elevated levels of apoptosis in fetal
kidneys of individuals carrying a PAX-2 mutation. End-
stage renal failure is commonly associated with this
syndrome, and one goal of this research was to
determine how PAX2 mutations could cause renal failure.
This was addressed firstly by characterizing the renal
phenotype in renal-coloboma syndrome patients. Renal
hypoplasia was the most common congenital abnormality
in a series of 29 patients, including 10 new patients
reported here.
Of the ten new patients identified with renal-
coloboma syndrome, nine were from a large 3-generation
Brazilian family transmitting a novel C -> T
substitution at position 1289 of PAX2, resulting in a
change from an arginine codon in exon 7 to a stop
codon. The mutation identified in the Brazilian family
is the most 3' mutation so far identified in PAX2, and
is the first report of a PAX2 mutation which leads
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directly to the introduction of a stop codon. If
expressed, the protein resulting from this mutant
allele is predicted to be truncated midway through the
partial homeodomain, and would result in loss of the
entire transactivation domain. PAX2 protein lacking
the transactivation domain is unlikely to be fully
functional, as studies have shown that this region in
the closely related PAX5 protein is required for
transcriptional activation of target genes. In the
tenth patient, a Japanese boy, we identified a 6619
insertion mutation of PAX2 exon 2. The 6619 insertion
mutation has previously been documented in other
patients (Favor, J. et al. (1996) Proc. Natl. Acad.
Sci. USA, 93, 1380-1387; Gavrielli, Y. et al. (1992) J.
Cell Biol., 119, 493-501). This patient also presented
with bilateral cryptorchidism, which has not previously
been described in renal-coloboma syndrome and may be
just a chance association. However, Pax2 is expressed
in the developing urogenital ridge, vas deferens, and
epidydimis (Dressler, G.R. et al. (1990) Development,
109, 787-795), and these structures are important in
the maturation of the genital tract. It is possible
that PAX2 mutations cause less obvious abnormalities of
the reproductive system in other patients with renal-
coloboma syndrome.
Four of the ten new patients in this report had
obvious renal hypoplasia; three others had clinical
evidence of renal insufficiency. When we reviewed
clinical data on the previously reported 19 cases of
proven human PAX2 mutations, we noted 13/19 with
obvious renal hypoplasia and all had evidence of renal
insufficiency or dysfunction. In at least two of the
three cases where renal biopsy had been performed in
childhood, there was striking atrophy of the proximal
and distal tubules, but nephron number could not be
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assessed. Renal hypoplasia was pronounced even in the
youngest patients in this study (patients 2 and 3), who
were 5 and 6 years old, suggesting that this is a
congenital anomaly.
Further studies to determine how PAX2 mutations
cause renal hypoplasia in human subjects were
impractical because it was not possible to obtain fetal
kidneys from patients with renal-coloboma syndrome.
lNeu
However, Pax2 mutant mice (Koseki, C. et al. (1992)
J. Cell Biol., 119, 1327-1333) harbor a mutation that
is identical to a mutation in approximately one third
of families with renal-coloboma syndrome. These mice
transmit a single base pair insertion amid a string of
seven guanidine residues (positions 613-619) in the
second exon of Pax2, which produces a frameshift and
presumably a null allele (Koseki, C. et al. (1992) J.
lNeu
Cell Biol., 119, 1327-1333). Pax2 mice have optic
nerve and kidney abnormalities, similar to humans with
renal-coloboma syndrome. Although the homozygous
mutants die within 24 hours of birth and are
phenotypically similar to ~~knockout" mice generated by
targeted homologous recombination, heterozygotes
survive and reach adulthood. The heterozygous mutant
mice therefore afford a useful animal model in which to
analyze fetal kidney defects associated with Pax2
mutations.
During normal kidney development Pax2 is first
detected along the nephrogenic cord at the sites from
which ureteric buds will emerge (Dressier, G.R. et al.
(1990) Development, 109, 787-795). Intense Pax2
expression persists in cells of the ureteric buds as
they invade the metanephric blastema to each side and
begin to undergo dichotomous branching (Dressier, G.R.
et al. (1990) Development, 109, 787-795, Eccles, M.R.
et al. (1992) Cell Growth & Diff., 3, 279-289).
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lNeu
Inspection of fetal Pax2 /+ mouse kidneys
demonstrated renal hypoplasia at an early (E15) stage
of development. This is apparently due to a decrease in
the rate of new nephron induction since the total
number of early epithelial structures (at the tips of
ureteric buds) and glomeruli (representing more
advanced nephrons) is strikingly reduced in mutant
kidneys. The nephrons that are formed, though reduced
in number, appear to have normal morphology. It can be
inferred, therefore, that arborization of the ureteric
bud is less complex in patients with heterozygous PAX2
mutations than in normal individuals. It should be
pointed out that even a modest reduction in the
efficiency of ureteric bud branching would reduce final
kidney size substantially, since the dichotomous
branching process is repeated many times during renal
development.
The downstream gene targets of PAX2 are largely
unknown, but appear to influence growth and branching
morphogenesis of the ureteric bud. For that reason we
examined the pattern of cell proliferation and
apoptotic cell death in embryonic kidneys of
lNeu
heterozygous Pax2 mutant mice. Cell division, as
assessed by PCNA immunohistochemistry and BrdU uptake,
was normal. However, apoptosis in the medullary
segments of the collecting duct (as assessed by TUNEL
assay) was strikingly increased at the time when Pax2
is maximally expressed in kidney development, and in
cells which normally express Pax2 (Dressier, G.R. et
al. (1990) Development, 109, 787-795). Indeed, the
amount and localization of the apoptosis correlated
temporally and spatially with the known expression
patterns of PAX2 (Dressier, G.R. et al. (1990)
Development, 109, 787-795, Eccles, M.R. et al. (1992)
Cell Growth & Diff., 3, 279-289), except that apoptosis
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was not observed in mesenchyme-derived epithelial
structures, whereas PAX2 is expressed in the
differentiating mesenchy::e and early mesenchyme-derived
structures. E15 mutant mice exhibited the greatest
levels of apoptosis, and at later stages when overall
PAX2 expression levels have declined (Dressier, G.R. et
al. (1990) Development, 109, 787-795), differences in
the rate of apoptosis in collecting ducts was less
lNeu
marked between heterozygous Pax2 /+ mutant and wild
type kidneys. Apoptosis is known to occur as part of
normal kidney development, but the rate of apoptosis
that we observed in E15 collecting ducts was
approximately 9-fold higher than in wild type offspring
at the same age. Since apoptotic cells are rapidly
cleared by phagocytosis, we can not ascertain whether
cell death is extensive enough to compromise
arborization of the ureteric bud. Conceivably, our
observation of increased apoptosis may reflect reduced
signaling by trophic factors influencing branching
morphogenesis as well as cell survival.
It is unclear why the apoptosis in the
heterozygous mutant kidneys was predominantly confined
to Wollfian duct-derived structures such as ureteric
buds and collecting ducts, even though Pax2 is also
expressed in mesenchymally-derived structures such as
comma- and S-shaped bodies. One possible explanation
arises from the observation that PaxB is co-expressed
with Pax2 in the differentiating mesenchyme-derived
structures, but not in ureteric buds or collecting
ducts. It is possible that, as has been described for
other Pax genes, expression of Pax8 was able to
compensate for heterozygous Pax2 mutations in the
mesenchymally-derived structures of kidneys from Pax2
mutant mice, and that this ultimately rescued these
cells from apoptosis.
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Our data do not determine whether PAX2 directly
regulates genes in apoptotic pathways during kidney
development. However, PAX2, PAX5 and PAX8 have all been
reported to inhibit p53 transcription, which can
promote apoptosis. Furthermore, transgenic mice
overexpressing wild type p53 have altered
differentiation of the ureteric bud, and have small
kidneys. In Pax5 knockout mice the B cells fail to
differentiate and undergo apoptotic cell death at an
early stage. Similarly, large-scale apoptosis of
photoreceptor precursors occurs in the eye discs of
Drosophila with the eyeless mutation (ey2) (homologous
to the mammalian Pax6 gene). These authors also found
no difference in BrdU uptake studies in wild type and
mutant eye discs. As in our studies, this suggests that
the mutation affects cell survival rather than
proliferation. Pax3 mutant mice were shown to have
enhanced apoptosis in the somatic mesoderm during
embryonic development, and treatment of
rhabdomyosarcoma cells lines with PAX3 antisense
oligonucleotides has been shown to result in apoptosis.
Unlike the above studies reporting apoptosis in
homozygous Pax mutant animals, our studies were carried
out on heterozygous mutant animals.
Others speculated that Pax2+/- mice might
develop renal hypoplasia due to a decreased rate of
cell proliferation in renal calyces and upper ureters.
However, our results suggest that the cause of the
small kidneys in renal-coloboma syndrome is due to
decreased cell survival in these structures rather than
decreased proliferation. The fact that the collecting
system is derived from the Wolffian duct, and that we
observed apoptosis at the earliest time-point examined
(E15), suggests that apoptosis could have occurred in
the Wolffian duct while the ureteric bud was sprouting
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from it. This notion would be consistent with, and may
help to explain the observed degeneration and lack of
caudal extension of the Wolffian duct in Pax2 null
mutant mice: In Pax2 mutants the lack of development
of, or dysplasia of the epidydimis, vas deferens,
seminal vesicles and ureters, each of which are derived
from the Wolffian duct, could also be caused by
enhanced apoptosis in the Wolffian duct and its
derivatives.
The effects of PAX2 haploinsufficiency on the
renal phenotype are similar to the developmental
effects of haploinsufficiency for other PAX genes. PAX3
mutations cause an autosomal dominant defect in
epidermal pigmentation, PAX6 mutations cause autosomal
dominant aniridia, and PAX8 mutations cause autosomal
dominant hypothyroidism. In each case, inactivation of
one allele is sufficient to interfere with normal organ
development. With regard to the mechanism of
haploinsufficiency, others have hypothesized that
monoallelic Pax gene expression may be related to the
semi-dominant effects of Pax gene mutations. We
analyzed the allelic expression pattern of Pax2 in lNeu
mutant kidneys both by RT-PCR, and by immunostaining.
In Pax2 mutant kidneys, immunostaining of Pax2 was
detected in virtually every cell in the ureteric buds.
Taken together with our RT-PCR data, these observations
suggest that Pax2 is biallelically transcribed in wild-
type fetal kidney, and that ureter and collecting duct
abnormalities produced by Pax2 haploinsufficiency
probably result from sensitivity to reduced gene dosage
in each cell. We can not rule out the possibility that
Pax2 is monallelically expressed at embryonic stages
earlier than E15, or in stem cells which were not
amenable to study by immunohistochemistry in E15
kidney.
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lNeu
In summary, the murine Pax2 mutation adds
much to our understanding of the human disease. We have
shown that renal hypoplasia is the most common
congenital renal anomaly in humans and mice with PAX2
mutations, and that in mice this phenotype is
associated with significantly enhanced apoptosis.
Furthermore, the Pax2 gene was found to be
biallelically expressed in mutant mice, therefore it
seems unlikely that the enhanced apoptosis was due to
random stochastic inactivation of Pax2 expression in
cells mono-allelically expressing Pax2. We conclude
from this work that haploinsufficiency of Pax2 leads to
small kidneys and renal failure because reduced Pax2
dosage compromises the survival of ureteric bud-derived
epithelial cells, and that this is associated with
reduced branching of the ureteric bud at an early stage
of development. Further studies are required to
determine if PAX2 is a primary determinant of cell
survival, or whether this effect is the consequence of
a default pathway of apoptosis in cells in which
development is disrupted.
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
