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J Am Soc Nephrol 15: 2548–2555, 2004
Feline Polycystic Kidney Disease Mutation Identified
in PKD1
LESLIE A. LYONS,* DAVID S. BILLER,† CAROLYN A. ERDMAN,*
MONIKA J. LIPINSKI,* AMY E. YOUNG,* BRUCE A. ROE,‡ BAIFANG QIN,‡ and
ROBERT A. GRAHN*
*Department of Population Health and Reproduction, School of Veterinary Medicine, University of California,
Davis, California; †Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University,
Manhattan, Kansas; and ‡Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma
Abstract. Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder in humans that causes the
formation of fluid-filled renal cysts, often leading to renal failure.
PKD1 mutations cause 85% of ADPKD. Feline PKD is autosomal dominant and has clinical presentations similar to humans.
PKD affects ~38% of Persian cats worldwide, which is ~6% of
cats, making it the most prominent inherited feline disease. Previous analyses have shown significant linkage between the PKD
phenotype and microsatellite markers linked to the feline homolog
for PKD1. In this report, the feline PKD1 gene was scanned for
causative mutations and a C⬎A transversion was identified at
c.10063 (human ref NM_000296) in exon 29, resulting in a stop
mutation at position 3284, which suggests a loss of ~25% of the
C-terminus of the protein. The same mutation has not been identified in humans, although similar regions of the protein are
truncated. The C⬎A transversion has been identified in the heterozygous state in 48 affected cats examined, including 41 Persians, a Siamese, and several other breeds that have been known
to outcross with Persians. In addition, the mutation is segregating
concordantly in all available PKD families. No unaffected cats
have been identified with the mutation. No homozygous cats have
been identified, supporting the suggestion that the mutation is
embryonic lethal. These data suggest that the stop mutation causes
feline PKD, providing a test to identify cats that will develop PKD
and demonstrating that the domestic cat is an ideal model for
human PKD.
Companion animal species such as the domestic cat are effective models for several inherited diseases. Cats and humans
show strong conservation of biology and anatomy and share
⬎30 homologous hereditary diseases (1,2). One inherited disease for which the cat is an exceptional animal model is
polycystic kidney disease.
Autosomal dominant polycystic kidney disease (ADPKD) is a
commonly inherited disorder in humans, with a frequency in the
general population of 1 in 1000 (3). Approximately 85% of
ADPKD cases are caused by mutations in the PKD1 gene (4,5),
located on human chromosome 16p13.3; the remaining 15% are
caused by mutations in the PKD2 gene, located on human chromosome 4q21–23 (6,7). ADPKD is characterized by the formation of fluid-filled cysts in the kidneys, and the average age of
onset is 40 yr, with ESRD occurring by age 60 in 50% of cases
(8). This suggests that ~4% of ESRD patients are a result of PKD.
Feline polycystic kidney disease is an inherited disease in
Persian and Persian-related cats. PKD in cats is characterized
by renal as well as hepatic and pancreatic cysts (9) and has an
autosomal dominant mode of inheritance (10,11). Approximately 38% of Persian cats in the United States (12) and
worldwide (13–17) are positive for PKD. Purebred cats represent ~20% of the cat population in the United States, and
Persian-type breeds constitute 80% of the cat fancy; hence,
PKD is the most prominent inherited feline disease.
A linkage analysis for feline PKD was performed by genotyping 43 feline-derived microsatellites in seven extended feline
pedigrees segregating for PKD (18). The results showed a significant linkage and no recombinants (Z ⫽ 5.83, ␪ ⫽ 0) between
feline PKD and the microsatellite marker FCA476 that is within
10 cR to the PKD1 gene on cat chromosome E3 (18).
A BAC clone that contains the feline PKD1 gene was identified
and submitted for sequencing at The University of Oklahoma’s
Advanced Center for Genome Technology. PCR amplification
and sequence analysis identified a C⬎A transversion causing a
stop codon (OPA) in exon 29 of the feline PKD1 gene. This
mutation has been identified in the heterozygous state in all 48
affected cats examined to date, including all affected Persians, a
Siamese, Ragdolls, domestic shorthairs, and several other breeds
that have been known to outcross with Persians, such as Exotic
Shorthair, Selkirk Rex, and Scottish Folds. The mutation is segregating consistently in the largest feline PKD pedigree and in
several individuals from all other available pedigrees. The causative mutation has not been observed in 33 unaffected cats, and no
homozygous affected cats have been identified, suggesting that
the mutation is embryonic lethal and is consistent with previous
Received May 5, 2004. Accepted July 18, 2004.
Correspondence to Dr. Leslie A. Lyons, 1114 Tupper Hall, Department of
Population Health & Reproduction, School of Veterinary Medicine, One
Shields Avenue, University of California, Davis, Davis, CA 95616. Phone:
530-754-5546; Fax: 530-752-4278; E-mail: [email protected]
1046-6673/1510-2548
Journal of the American Society of Nephrology
Copyright © 2004 by the American Society of Nephrology
DOI: 10.1097/01.ASN.0000141776.38527.BB
J Am Soc Nephrol 15: 2548–2555, 2004
data (18). These data suggest that the stop mutation causes feline
PKD and that a DNA test is now possible to identify cats that will
develop PKD in the future. Along with the similar clinical presentation, these data support the use of the domestic cat as a model
for human PKD. The cat has the same mode of inheritance, a
mutation in the PKD1 gene; the affected cats have only the
heterozygous state, and they can be used for long-term drug and
potentially gene therapy trials.
Cat PKD Mutation Identified
2549
Capillary DNA Sequencer. The resulting sequence data were transferred to a Sun Workstation Cluster, where it was base-called and
assembled using the Phred and Phrap programs (24,25). Overlapping
sequences and contigs were analyzed using Consed (26).
Fluorescence in situ hybridization (FISH) of the PKD1 BAC
clone was performed on mitotic spreads of feline chromosomes
using standard methods. Labeling procedures and probe concentrations were the same as described previously (27). Cot-1 DNA
was substituted in the hybridization solution with a twofold amount
of cat genomic DNA.
Materials and Methods
All animal experimentation described in this article was conducted
in accord with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals.
Sample Identification
Samples from cats that represent families segregating with PKD
were collected from five feline PKD ultrasound screening clinics that
were held at the University of California, Davis, School of Veterinary
Medicine between June 2000 and September 2002. Cats of 10 mo of
age or older were determined to be affected or normal by the visualization of singular bilateral or multiple unilateral cysts using ultrasonography. The same two board-certified radiologists (D.A.B. and
Dr. E. Herrgesell of the University of California, Davis, School of
Veterinary Medicine) scanned all cats. Details regarding the disease
criteria and the pedigrees developed from these clinics have been
previously described (18). Forty-eight affected and 33 normal cats
were analyzed. Representatives from each pedigree were genotyped
for the PKD mutation, including 41 affected and 26 unaffected Persians. One PKD-affected cat and one normal cat from other breeds,
including Siamese, Siberian, Exotic Shorthair, Domestic Shorthair,
Selkirk Rexes, Scottish Folds, and Ragdolls, were identified by the
authors (D.J.B.) and/or from the University of California, Davis,
ultrasound screening clinics and were also analyzed.
BAC Clone Sequencing
The sequence of the feline BAC clone (GenBank accession no.
AC145332.26) that contains the feline PKD1 homolog was obtained
using standard BAC isolation, shotgun sequencing, and finishing
strategies as described previously (19 –23). Primers from exon 17 that
had an 8-bp overlap in the 3' portion of their sequence were constructed. Thus, when amplified, they produced a product of 40 bp that
was used as a probe to isolate the BAC clone that contains the feline
PKD1 gene. Commonly termed “overgo” primers, the sequences for
these primers were PKD1F tgcccattgtgtccttggagtgtg and PKD1R tgtgccttgcaggacacacactcc. Briefly, 50 ␮g of purified BAC DNA was
randomly sheared and made blunt-ended. After kinase treatment and
gel purification, fragments in the 1 to 3 kb range were ligated into
SmaI-cut, bacterial alkaline phosphatase (BAP)-treated pUC18 (Pharmacia) and transformed by electroporation into Escherichia coli,
strain XL1BlueMRF’ (Stratagene, La Jolla, CA). A random library of
~2500 colonies were picked from the transformation and grown in
Terrific Broth (TB) medium supplemented with 100 ␮g of ampicillin
for 14 h at 37°C with shaking at 250 rpm, and the sequencing
templates were isolated by a cleared lysate-based protocol. Sequencing reactions were performed as described previously using TaqDNA
polymerase with the Amersham ET Fluorescence-labeled terminators
(19 –22). The reactions were incubated for 60 cycles in a Perkin-Elmer
Cetus DNA Thermocycler 9600, and after removal of unincorporated
dye terminators by ethanol precipitation, the fluorescence-labeled
nested fragment sets were resolved by electrophoresis on an ABI 3700
PKD1 Sequence Analysis
Sequence of the PKD1-containing BAC clone (GenBank accession no. AC145332.26) was aligned to the human (GenBank accession
no. AC009065.8), dog (GenBank accession no. AY102170.1), and
mouse (GenBank accession no. AC132367.3) PKD1 sequences to identify potential intron/exon boundaries for the cat using the software PipMaker (28) (Figure 1). Primers were developed in intronic regions for
amplification of complete exons using the software Primer3 (http://
frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (29) and NetPrimer
(http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.
html). Primers and the GenBank accession numbers for the exons
analyzed are presented in Table 1. Primers (MWG Biotech, High
Point, NC) were used to amplify PKD-negative control cat DNA.
Each primer was tested in the cat as described previously (30) on
a Stratagene 96-well temperature gradient Robocycler. The amplified
products were separated on 1.8% agarose gels at 100 Vhr. Gels were
visualized by UV exposure after ethidium bromide staining and photodocumented using the Alpha Imaging System (Alpha Innotech, San
Leandro, CA). A positive optimization of the primers produced a single
PCR product that was excised from the gel and purified using the Qiagen
gel extraction column (Qiagen, Valencia, CA), or PCR products were
purified directly using the Qiagen PCR clean up kit. Purified products
were sequenced directly in both directions using the ABI Dye Terminator
Sequencing chemistry v3.1 (Applied Biosystems, Foster City, CA). Sequencing reactions were separated on an ABI 377 DNA Analyzer, and
the DNA contig sequence was assembled using the Sequencer Software
package (Gene Codes, Ann Arbor, MI). Integrity of the sequence contig
was confirmed by visual inspection and verified to be the correct gene by
comparison with sequences in GenBank using BLAST (31).
Genotyping
DNA from 48 affected and 33 normal cats was isolated from
white cells by standard phenol/chloroform techniques. EDTA anticoagulated blood was collected by venipuncture at the PKD clinics
or sent by private clinicians. PKD exon products were amplified
by PCR from genomic DNA of two normal and two affected cats
using the optimized primers (Table 1). Individual exons were
amplified independently in feline DNA samples using optimal
PCR conditions on a Stratagene 96-well temperature gradient
Robocycler. Approximately 12.5 ng of DNA was used per PCR
reaction. Reaction conditions for each primer pair were as follows:
~1 pmol of each forward and reverse primer, 1.25 mM dNTP, 1.75
mM MgCl2, 1⫻ PCR buffer II, and 0.375U of Amplitaq (Applied
Biosystems) polymerase in 10 ␮l reaction volumes. Cycling parameters included an initial 3 min denaturation at 94°C followed by
35 cycles of 1 min denaturation at 94°C, annealing for 1 min
at 58°C, and a 72°C extension for 1 min. The cycling parameters
were followed by a final extension at 72°C for 10 min. Products
were generated, visualized, gel-extracted, purified, and sequenced
as described above and analyzed for mutations associated with
2550
Journal of the American Society of Nephrology
J Am Soc Nephrol 15: 2548–2555, 2004
Figure 1. Schematic diagram of the feline PKD1 gene as suggested from the BAC clone sequence. Intron/exon boundaries are predicted by
comparison with human, mouse, and dog sequence as determined with the program PipMaker (28). The y axis represents nucleotide identity
of the cat to the human sequence, and the x axis represents nucleotide position in the gene. The exon number is represented above each exon
symbol. □, simple repeat; 䡵, UTR; 3, exon; 䡵, gene; , CpG/GpCⱖ0.75; , CpG/GpCⱖ0.60.
Table 1. PCR analysis of PKD1 in the domestic cat
Exon
Exon Size
(bp)
Product
Size (bp)
Forward Primer 5⬘-3⬘
Reverse Primer 5⬘-3⬘
GenBank
Accession No.
6
14
15
21–22
22–23
23
24
29
30
37
38
183
133
3619
153,145
145,630
629
156
210
126
194
139
415
271
533
900
349
572
421
558
376
302
267
cacctctcctgatcctcctc
cggacaccactctttcactc
catcccccatgtcaaaagt
gcccagatacgcaagaacat
ctgaagaagacgctgcacaa
gaaagacacctgctcaccaa
acctactcccacaggaaacc
caggtagacgggatagacga
tcgtctcgaccttctgcc
cagacacgggacaggaga
gacaagatcgagatgggatg
gccacctacagtattgtgtcttt
actccagcctgctcattgt
agcacggggttaggtcat
tgtgatgttgaggatgctgtc
ggagcgcatgaggatacac
ggccctcatgtgtatcctc
ggaacgaggcaacagtga
ttcttcctggtcaacgactg
cctcgtctgcctcttcct
ctcaaggtgagtgggatgtt
cacactgggattggctga
No variants
No variants
No variants
AY660021
AY686652
No variants
AY612846
AY612847
No variants
AY612848
AY612849
PKD. Sequences generated from each exon were aligned (DNAStar, Madison, WI) with wild-type cat sequence to identify possible
causative mutations for the observed phenotype. When polymorphisms were detected, sequence data were translated to determine
whether the mutation resulted in an amino acid change.
ducing two fragments of 316 and 243 bp. Approximately 5 ␮l of
amplification product was digested with 10 U of MLY1 (New England
Biolabs, Beverly, MA) in a 10 ␮l reaction that contained 1⫻ NE
Buffer 4 at 37°C for 3 h followed by inactivation of the enzyme at
65°C for 10 min. The complete digestion reaction was analyzed on 1.8
to 2% agarose gels as described above.
RFLP Analysis
Once the mutation was identified, 46 affected and 31 normal cats
were screened for the PKD mutation using RFLP typing on agarose
gels. The amplification product for exon 29 is 559 bp. The identified
mutation causes a restriction enzyme site alteration for MLY1, pro-
Results
The sequenced BAC clone (GenBank accession no.
AC145332.26) is in eight contigs that cover 167 kb. The region
J Am Soc Nephrol 15: 2548–2555, 2004
Cat PKD Mutation Identified
2551
Table 2. Sequence analysis of feline PKD1 compared with other species
Size (bp)
% Sequence Identity to Species
% Protein Identity to Species
Exon/Intron
Human
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
424/16096
72/121
72/268
170/213
672/118
184/435
221/188
116/410
127/366
248/452
756/877
132/197
176/314
134/468
3620/219
150/934
144/127
280/93
214/66
160/390
153/679
145/602
630/295
157/180
253/124
196/1494
171/86
144/94
211/90
127/1659
117/87
53/224
185/77
94/2937
119/78
203/72
195/450
140/361
113/291
142/140
126/183
175/248
291/75
135/83
306/90
1485
Cat
na/14150
72/109
72/280
170/na
672/94
184/1093
221/413
116/413
127/314
248/434
755/806
132/194
176/195
134/548
3620/156
150/1026
204/108
280/75
214/72
172/336
153/691
145/699
621/306
157/247
254/109
196/1020
171/80
144/89
211/89
127/766
117/103
53/158
173/106
94/2565
119/75
203/75
195/369
140/620
113/205
148/154
126/269
175/135
291/93
136/80
306/95
1466
Dog
Human
NA
91.7
88.9
88.8
86.9
94.0
88.2
90.5
86.6
75.0
83.0
91.7
86.4
83.2
76.0
90.7
86.8
92.9
90.2
84.3
84.3
86.9
86.6
83.4
86.6
88.8
95.9
93.8
91.0
91.3
88.0
90.6
89.6
79.8
78.2
88.2
91.3
87.1
91.2
79.1
94.4
90.3
86.9
84.4
86.9
81.2
NA
90.3
94.4
82.9
79.3
91.8
82.4
81.9
73.2
64.9
77.6
88.6
84.7
80.6
80.4
84.7
85.4
86.4
85.5
73.8
73.9
84.8
80.7
79.0
81.8
81.1
88.3
88.9
85.3
87.4
88.0
83.0
84.4
71.3
80.7
85.7
87.7
84.3
90.3
76.8
89.7
88.6
82.8
77.0
82.0
66.7
that contains the PKD1 gene is represented by two contigs that
are separated in intron 4. The 5' region of exon 1 is not
complete, with an estimated 278 bp not represented in the
assembled sequence. A schematic of the feline PKD1 gene is
Mouse
Dog
Human
Mouse
NA
77.8
88.9
72.4
74.4
84.8
78.4
59.5
73.2
45.3
67.3
81.8
80.7
73.1
71.6
80.0
78.5
77.9
68.7
58.1
67.3
84.1
78.1
83.4
76.7
80.6
78.9
89.6
80.6
77.2
71.9
76.0
79.2
70.2
68.1
82.8
86.2
85.0
83.2
76.8
83.3
77.7
71.8
77.0
70.3
67.8
NA
95.7
91.3
87.5
83.4
95.0
94.5
92.1
88.1
67.1
84.8
97.7
89.7
76.7
87.3
96.0
85.4
97.8
97.1
86.0
84.3
89.6
87.3
80.4
90.5
92.3
100.0
95.7
90.5
92.9
84.6
88.2
87.7
63.3
59.0
86.6
98.5
95.7
94.6
68.8
97.6
93.1
87.6
80.0
95.1
85.5
NA
91.3
95.7
87.5
74.9
95.0
84.9
81.6
76.2
57.3
80.8
90.9
87.9
75.0
79.6
84.0
79.2
92.5
91.4
71.7
68.6
81.2
83.4
84.3
86.9
78.5
87.5
93.6
83.9
85.7
84.6
88.2
78.9
50.0
66.7
86.6
96.9
87.0
91.9
54.3
85.4
87.9
81.4
80.0
83.3
78.7
NA
73.9
87.0
69.6
67.3
86.7
86.1
60.5
73.8
41.0
73.2
93.2
87.9
75.0
75.7
88.0
72.9
86.0
81.4
71.7
60.0
85.4
81.0
80.4
84.5
86.2
89.3
95.7
78.7
78.6
60.5
75.0
77.2
46.7
46.2
85.1
95.4
89.1
91.9
58.7
85.4
93.1
69.1
77.8
78.4
65.4
presented in Figure 1, which predicts exon sizes in the genomic
BAC clone and sequence identity to the annotated human
mRNA sequence. Intron and exon sizes as well as estimates of
sequence and protein identity to human, mouse, and dog are
2552
Journal of the American Society of Nephrology
presented in Table 2. Over all exons, the cat sequence is most
similar to dog (83.66%; range, 75.0 to 94.4%), least similar to
mouse (73.44%; range, 45.3 to 89.6%), and intermediately
similar to humans (80.02%; range, 64.9 to 94.4%). Exon 10 has
the lowest similarity with the cat as compared with each
species. The similarity of cat sequence to that from any second
species (mouse, human, or dog) varies between exons. One
would expect the cat always to be most similar to the dog, a
fellow carnivore, but this was not the case. The predicted
mRNA and protein sequence is provided in a Supplemental
figure. FISH of the PKD1 BAC clone does not suggest duplication of the feline PKD1 gene (Figure 2), and the feline gene
is located on cat chromosome E3. Nine of 46 PKD1 exons
were scanned for mutations by direct sequencing. A C⬎A
transversion at c.10063 (human ref NM_000296) in exon 29
resulting in a C3284X protein change was identified, which is
an OPA stop codon that should cause a loss of ~25% of the
protein (Figure 3). The mutation causes a unique RFLP site in
the amplification product of exon 29. An example of the RFLP
typing is presented in Figure 4. A total of 48 affected and 33
unaffected cats were scanned for this stop mutation. Ten cats
were confirmed by sequence analyses and all cats by RFLP.
All 48 affected cats had the stop mutation, including the
non-Persian cats. None of the 33 unaffected cats was identified
with the mutation. Pedigree analysis of feline PKD family 5
(18) showed complete co-segregation of the stop mutation with
the disease phenotype. None of the 48 affected cats was found
to be homozygous for the mutation.
Four of the nine exons (exons 6, 14, 15, 23, 24, 29, 30, 37,
38) and several intron regions had nucleotide variants as identified between two sequenced Persian cats and the PKD1
sequence from the BAC clone (Table 1). One Persian was
affected with PKD; thus, the sequence comparison represents
three normal alleles. Each identified variant was homozygous
in the two Persian cats, except for a mutation in exon 29. Ten
variants were identified, but only four were in translated regions of the exons. None was identified at exon/intron splice
J Am Soc Nephrol 15: 2548–2555, 2004
sites. Two of the four variants produced silent mutations and
were homozygous in the two Persian cats sequenced. One
mutation, a C⬎T transition at position 127 of exon 38, caused
an amino acid change, but both amino acids are hydrophobic
and it is not anticipated that this substitution alters the protein
conformation. One primer set amplified exons 21 and 22 and
the intervening intron 21, and another set amplified exons 22
and 23 and the intervening intron 22 (Table 1). Both cat introns
are shorter than the corresponding human intron (Table 2), and
no sequence data from the BAC clone and/or amplified
genomic DNA supported the presence of polypyrimidine tracts.
Discussion
Domestic cats are effective models for several inherited
diseases and should be used to develop better drug and gene
therapies for PKD. Feline PKD is an inherited disease in
Persian and Persian-related cats. As with humans, PKD in cats
is characterized by renal as well as hepatic and pancreatic cysts
(9) and has an autosomal dominant mode of inheritance (10).
Approximately 37% of Persian cats worldwide (14) are PKD
affected. Only 20% of the cat population in the United States
is represented by purebred cats, but of purebreds, Persians and
Persian-derived breeds constitute ~80% of the cat fancy (32).
This suggests that ~6% of the cat population in the United
States has PKD, making it the most prominent inherited feline
disease.
Our previous linkage analyses strongly implicated PKD1 as
the causative gene for feline PKD (18). Each newly identified
family for human PKD has generally been found to be a novel
mutation in the PKD1 gene. Mutations are found throughout
the gene, with no single mutation being highly prevalent in the
population. Because no particular mutation or region of the
PKD1 gene in humans is highly prevalent for mutations, identification of the feline PKD mutation could have entailed the
complete sequencing of the feline homolog from an affected
cat. Once the draft sequence of the feline BAC clone containing PKD1 was obtained, we began a systematic scan for
Figure 2. Localization of the feline
PKD1 gene by fluorescence in situ
hybridization to domestic cat chromosome E3q13. Presented is a mitotic spread of feline chromosomes
from lymphocytes with the hybridization of the feline PKD1 BAC
clone (a; arrows). Hybridization
signals are observed as red dots on
both E3 chromosomes. Each dot
represents the signal of a single
chromatid. (b) The inverted DAPIbanding pattern of the same
metaphase. Feline chromosome
E3 is the smallest metacentric cat
chromosome and is easily distinguished by its morphology and
DAPI-banding pattern.
J Am Soc Nephrol 15: 2548–2555, 2004
Cat PKD Mutation Identified
2553
Figure 3. (a) DNA alignment of PKD1 exon
29 from mouse, human, dog, and cat. Both
wild-type and mutant sequences are included
from the cat. Boldface in cat sequence indicates the transversion observed in polycystic
kidney disease (PKD)-affected cats causing a
change in the amino acid translation resulting
in an OPA stop codon. (b) Protein alignment
of PKD1 exon 29 from mouse, human, dog,
and cat. Both wild-type and mutant translations are included from the cat. Boldface X in
the cat sequence indicates the OPA stop
codon observed in PKD-affected cats.
Figure 4. RFLP typing for the feline PKD mutation. Amplification
products of exon 29 were digested with the restriction enzyme MLY1.
The 559-bp product is digested into two fragments of 316 and 243 bp.
All cats affected with PKD produced the wild-type 559-bp fragment
and the digested fragments. A 1-kb⫹ marker is in lanes A and F. Lanes
B and E are unaffected cats, and lanes C and D are affected cats.
mutations. A nucleotide transversion causing a stop codon was
identified in exon 29 that suggests a truncation of the protein
with a loss of 25% of its C-terminus. Thus, this feature is a very
strong candidate for the causative mutation resulting in the
PKD phenotype.
In addition, concordant segregation of the mutation with the
disease in a large cat family segregating for PKD supports the
stop mutation as causative for feline PKD. A different mutation
still possibly could cause feline PKD but would have to be in
strong linkage disequilibrium with this stop codon. In addition,
the stop codon mutation is consistent with the microsatellite
haplotype that shows complete linkage to the PKD phenotype
(data not shown).
The feline PKD1 gene is represented by two contigs, with
the 5' region of exon 1 and intron 4 not yet represented by
sequence. A majority of feline exon and intron sizes are estimated from the BAC clone sequence. In humans, exon 1 is 424
bp; thus, ~141 amino acids cannot be compared with the cat.
Intron 4 is 213 bp in humans, and this entire intron may not yet
be represented in the cat sequence. Over all exons, the cat
sequence is most similar to dog, followed by human, and least
similar to mouse, which is consistent with the evolutionary
relationship of the species. Exon 10 is the least similar for all
species, as compared with the cat.
There are two distinctive features found in the human PKD1
gene that distinguish it from the homolog in mice and dogs
(33–36): (1) the presence of polypyrimidine tracts in intron 21
(~2.5 kb) and intron 22 (~600 bp) and (2) the presence of
several replicates of a 13.5-kb region of the 5' portion of the
gene on human chromosome 16. Neither feature is present in
the cat. The genomic amplifications of introns 21 and 22 from
both normal and affected cats are consistent with the size
determined by the feline BAC clone sequencing. Introns 21
and 22 both are smaller in cats than in humans. When humanderived primers were used to amplify a region that consisted of
a majority of exon 21, exon 22, and the intervening intron 21,
this region is ~950 bp in cats and could not be amplified from
genomic human DNA, suggesting that the 2.5-kb tract could
not be amplified by standard PCR. The polypyrimidine tract is
2554
Journal of the American Society of Nephrology
not represented in the human reference sequence for PKD1 but
has been demonstrated elsewhere and confirmed by long-PCR
techniques (34). Intron 22 is only 349 bp as compared with 602
bp in humans. Thus, the polypyrimidine features are not found
in cats.
Likewise, three aspects of our data suggest that the 5' portion
of the feline PKD1 gene homolog is not replicated as in
humans. For the cats sequenced, heterozygous sequence was
not identified within the exons and introns that correspond to
the region duplicated in humans. Pseudogenes acquire mutations rapidly; thus, it would be anticipated that some variants
would be detected. FISH of the PKD1 BAC clone does not
suggest duplication and was located on feline chromosome E3,
which is as predicted by genetic mapping (37) and chromosome painting (38) and is the region homologous to human
chromosome 16. In addition, because this region is not replicated in both mice and dogs, it is more likely that this replication event is found in humans and/or primates and is not an
ancestral event for mammals.
Only the Persian and Persian-derived cat breeds (Exotic Shorthairs and Himalayans) have been recognized to have a high
frequency of PKD. Other breeds—Ragdolls, Scottish Folds, and
Selkirk Rexes— have either purposely or accidentally bred to
Persians. We hypothesize the PKD mutation in the other breeds to
be identical by descent with the Persian mutation. All affected cats
from the other breeds have the identical mutation as the Persians.
Because a majority of human cases are de novo, more extensive
sequence analyses are required to differentiate de novo, identical
by state mutations from mutations that are identical by descent
within various cat breeds. This disease is occurring within a closed
breed, suggesting that identity by descent and disease homogeneity is expected. Persians, however, are one of the oldest and most
popular breeds, having a large population that is dispersed
throughout the world. All cases analyzed here represent cats from
the United States; thus, more extensive surveys should be conducted to validate the causative mutation in different regions of
the world.
Further investigation into the cause of PKD will be valuable
for feline health as well as provide insights into human ADPKD. As with humans, cats have a wide range of disease
progression and severity; thus, other genetic and environmental
factors could influence disease progression (39 – 42). Currently, the disease is highly prevalent in the cat population,
making the identification of both severe, early-onset cases and
mild, late-onset cases feasible. This could lead to the identification of genetic modifiers. As cats have similar clinical presentations, therapies that are under development for epidermal
growth factor receptor (EGFR) could be tested for efficacy in
the cat, before use in humans (43– 47).
Persian and Persian-related cats should be screened for PKD
by ultrasound before they are bred (47). Although breeders are
advised not to breed two positive cats, they are often bred for
several unrelated reasons: (1) clinical signs have not yet appeared; (2) many breeders are still unaware of the disease; (3)
ultrasound is either unavailable or cost-prohibitive; (4) breeding decisions are made before adequate accuracy of diagnosis;
and (5) the disease is highly prevalent; thus, many catteries
J Am Soc Nephrol 15: 2548–2555, 2004
could lose ~40% of their breeding population. A genetic test
for feline PKD will provide breeders with an efficient and
accurate means to selectively breed their cats and remove PKD
from the population. Because PKD has been found in other cat
breeds related to Persians, the incidence of PKD in these
breeds should be evaluated.
Acknowledgments
Funding has been provided to L.A.L. by the Winn Feline Foundation, the Waltham Foundation, and the George and Phyllis Miller
Feline Health Fund, Center for Companion Animal Health, School of
Veterinary Medicine, University of California, Davis, and to B.A.R.
from National Institutes of Health, National Human Genome Research
Institute Grant HG002153.
We are grateful to the cat breeders who participated in the PKD
screening clinics and provided samples for analysis and the FISH data
from Dr. Lutz Froenicke. Patent pending: UC Case No. 2004-447.
References
1. Migaki G: Compendium of Inherited Metabolic Diseases in
Animals, New York, A.R. Liss, 1982
2. Nicholas FW, Brown SC, Le Tissier PR: Mendelian Inheritance
in Animals (MIA), Sydney, Australia, Department of Animal
Science, University of Sydney, 1998
3. Dalgaard OZ: Bilateral polycystic disease of the kidneys; a
follow-up of 284 patients and their families. Dan Med Bull 4:
128 –133, 1957
4. Consortium TEPKD: The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on
chromosome 16. Cell 77: 881– 894, 1994
5. Consortium TIPKD: Polycystic kidney disease: The complete structure of the PKD1 gene and its protein. Cell 81: 289 –298, 1995
6. Kimberling WJ, Kumar S, Gabow PA, Kenyon JB, Connolly CJ,
Somlo S: Autosomal dominant polycystic kidney disease: Localization of the second gene to chromosome 4q13-q23. Genomics
18: 467– 472, 1993
7. Peters DJ, Spruit L, Saris JJ, Ravine D, Sandkuijl LA, Fossdal R,
Boersma J, van Eijk R, Norby S, Constantinou-Deltas CD, et al.:
Chromosome 4 localization of a second gene for autosomal
dominant polycystic kidney disease. Nat Genet 5: 359 –362, 1993
8. Gabow PA: Autosomal dominant polycystic kidney disease.
N Engl J Med 329: 332–342, 1993
9. Eaton KA, Biller DS, DiBartola SP, Radin MJ, Wellman ML:
Autosomal dominant polycystic kidney disease in Persian and
Persian- cross cats. Vet Pathol 34: 117–126, 1997
10. Biller DS, Chew DJ, DiBartola SP: Polycystic kidney disease in a
family of Persian cats. J Am Vet Med Assoc 196: 1288 –1290, 1990
11. Biller DS, DiBartola SP, Eaton KA, Pflueger S, Wellman ML,
Radin MJ: Inheritance of polycystic kidney disease in Persian
cats. J Hered 87: 1–5, 1996
12. Cooper B: Autosomal dominant polycystic kidney disease in
Persian cats. Feline Prac 28: 20 –21, 2000
13. Barthez PY, Rivier P, Begon D: Prevalence of polycystic kidney
disease in Persian and Persian related cats in France. J Feline
Med Surg 5: 345–347, 2003
14. DiBartola S: Autosomal dominant polycystic kidney disease. In:
Proceedings of the 18th Annual Veterinary Medical Forum of the
American College of Veterinary Internal Medicine, Seattle,
American College of Veterinary Internal Medicine, 2000, pp
438 – 440
J Am Soc Nephrol 15: 2548–2555, 2004
15. Cannon MJ, MacKay AD, Barr FJ, Rudorf H, Bradley KJ,
Gruffydd-Jones TJ: Prevalence of polycystic kidney disease in Persian cats in the United Kingdom. Vet Rec 149: 409 – 411, 2001
16. Barrs VR, Gunew M: Prevalence of autosomal dominant polycystic kidney disease in Persian cats and related-breeds in Sydney and Brisbane. Aust Vet J 79: 257–259, 2001
17. Beck C, Lavelle RB: Feline polycystic kidney disease in Persian
and other cats: A prospective study using ultrasonography. Aust
Vet J 79: 181–184, 2001
18. Young A, Biller D, Herrgesell E, Roberts H, Lyons L: Feline
polycystic kidney disease linkage to the PKD1 region. Mamm
Genome (in press), 2004
19. Roe BA: Shotgun Library Construction for DNA Sequencing,
Totowa, Humana Press, 2004
20. Bodenteich A, Chissoe S, Wang YF, Roe BA: Shotgun Cloning as
the Strategy of Choice to Generate Templates for High-Throughput
Dideoxynucleotide Sequencing, London, Academic Press, 1993
21. Chissoe SL, Bodenteich A, Wang YF, Wang YP, Burian SW,
Dennis C, Crabtree J, Freeman A, Iyer K, Jian L, Ma Y, McLaury
HJ, Pan HQ, Sharan O, Toth S, Wong Z, Zhang G, Heisterkamp N,
Groffen J, Roe BA: Sequence and analysis of the human ABL gene,
the BCR gene, and regions involved in the Philadelphia chromosomal translocation. Genomics 27: 67– 82, 1995
22. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A
Laboratory Manual, New York, Cold Spring Harbor Laboratory
Press, 1989
23. McPherson JD, Marra M, Hillier L, Waterston RH, Chinwalla A,
Wallis J, Sekhon M, Wylie K, Mardis ER, Wilson RK, Fulton R,
Kucaba TA, Wagner-McPherson C, Barbazuk WB, Gregory SG,
Humphray SJ, French L, Evans RS, Bethel G, Whittaker A,
Holden JL, McCann OT, Dunham A, Soderlund C, Scott CE,
Bentley DR, Schuler G, Chen HC, Jang W, Green ED, Idol JR,
Maduro VV, Montgomery KT, Lee E, Miller A, Emerling S,
Kucherlapati, Gibbs R, Scherer S, Gorrell JH, Sodergren E,
Clerc-Blankenburg K, Tabor P, Naylor S, Garcia D, de Jong PJ,
Catanese JJ, Nowak N, Osoegawa K, Qin S, Rowen L, Madan A,
Dors M, Hood L, Trask B, Friedman C, Massa H, Cheung VG,
Kirsch IR, Reid T, Yonescu R, Weissenbach J, Bruls T, Heilig R,
Branscomb E, Olsen A, Doggett N, Cheng JF, Hawkins T, Myers
RM, Shang J, Ramirez L, Schmutz J, Velasquez O, Dixon K,
Stone NE, Cox DR, Haussler D, Kent WJ, Furey T, Rogic S,
Kennedy S, Jones S, Rosenthal A, Wen G, Schilhabel M, Gloeckner G, Nyakatura G, Siebert R, Schlegelberger B, Korenberg
J, Chen XN, Fujiyama A, Hattori M, Toyoda A, Yada T, Park
HS, Sakaki Y, Shimizu N, Asakawa S, Kawasaki K, Sasaki T,
Shintani A, Shimizu A, Shibuya K, Kudoh J, Minoshima S,
Ramser J, Seranski P, Hoff C, Poustka A, Reinhardt R, Lehrach
H: A physical map of the human genome. Nature 409: 934 –941,
2001
24. Ewing B, Green P: Base-calling of automated sequencer traces
using phred. II. Error probabilities. Genome Res 8: 186 –194, 1998
25. Ewing B, Hillier L, Wendl MC, Green P: Base-calling of automated sequencer traces using phred. I. Accuracy assessment.
Genome Res 8: 175–185, 1998
26. Gordon D, Abajian C, Green P: Consed: A graphical tool for
sequence finishing. Genome Res 8: 195–202, 1998
27. Frönicke L, Anderson LK, Wienberg J, Ashley T: Male mouse
recombination maps for each autosome identified by chromosome painting. Am J Hum Genet 71: 1353–1368, 2002
28. Schwartz S, Zhang Z, Frazer K, Smit A, Riemer C, Bouck J, Gibbs R,
Hardison R, Miller W: PipMaker: A web server for aligning two
genomic DNA sequences. Genome Res 10: 577–586, 2000
Cat PKD Mutation Identified
2555
29. Rozen S, Skaletsky HJ: Primer3 on the WWW for General Users
and for Biologist Programmers, Totowa, Humana Press, 2000
30. Lyons LA, Laughlin TF, Copeland NG, Jenkins NA, Womack JE,
O’Brien SJ: Comparative anchor tagged sequences (CATS) for integrative mapping of mammalian genomes. Nat Genet 15: 47–56, 1997
31. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic
local alignment search tool. J Mol Biol 215: 403– 410, 1990
32. CFA: Cat Fanciers’ Association registration totals by color and
breed—2003, and 1/1/58 to 12/31/03. Cat Fanciers’ Almanac
20:72– 86, 2004
33. Piontek KB, Germino GG: Murine Pkd1 introns 21 and 22 lack
the extreme polypyrimidine bias present in human PKD1. Mamm
Genome 10: 194 –196, 1999
34. Afzal AR, Jeffery S: Amplification of a 13.5-Kb region of the
PKD1 gene containing the 2.5-Kb polypyrimidine tract in intron
21 facilitates mutation detection in this gene. Genet Test 5:
57–59, 2001
35. Watnick TJ, Gandolph MA, Weber HNH, Germino G: Gene
conversion is a likely cause of mutation in PKD1. Hum Mol
Genet 7: 1239 –1243, 1998
36. Watnick TJ, Piontek KB, Cordal TM, Weber H, Gandolph MA,
Qian F, Lens XM, Neumann HP, Germino G: An unusual pattern
of mutation in the duplicated portion of PKD1 is revealed by use
of a novel strategy for mutation detection. Hum Mol Genet 6:
1473–1481, 1997
37. Murphy WJ, Sun S, Chen Z, Yuhki N, Hirschmann D, MenottiRaymond M, O’Brien SJ: A radiation hybrid map of the cat
genome: implications for comparative mapping. Genome Res 10:
691–702, 2000
38. O’Brien SJ, Wienberg J, Lyons LA: Comparative genomics:
Lessons from cats. Trends Genet 13: 393–399, 1997
39. Bogdanova N, Dworniczak B, Dragova D, Todorov V, Dimitrakov D, Kalinov K, Hallmayer J, Horst J, Kalaydjieva L: Genetic
heterogeneity of polycystic kidney disease in Bulgaria. Hum
Genet 95: 645– 650, 1995
40. Parfrey PS, Davidson WS, Green JS: Clinical and genetic epidemiology of inherited renal disease in Newfoundland. Kidney
Int 61: 1925–1934, 2002
41. Tahvanainen P, Tahvanainen E, Reijonen H, Halme L, Kaariainen H, Hockerstedt K: Polycystic liver disease is genetically
heterogeneous: Clinical and linkage studies in eight Finnish
families. J Hepatol 38: 39 – 43, 2003
42. Magistroni R, Manfredini P, Furci L, Ligabue G, Martino C,
Leonelli M, Scapoli C, Albertazzi A: Epidermal growth factor
receptor polymorphism and autosomal dominant polycystic kidney disease. J Nephrol 16: 110 –115, 2003
43. Davis ID, MacRae Dell K, Sweeney WE, Avner ED: Can progression of autosomal dominant or autosomal recessive polycystic kidney disease be prevented? Semin Nephrol 21: 430 – 440,
2001
44. Avner ED, Woychik RP, Dell KM, Sweeney WE: Cellular pathophysiology of cystic kidney disease: Insight into future therapies.
Int J Dev Biol 43: 457– 461, 1999
45. Sweeney WE, Futey L, Frost P, Avner ED: In vitro modulation
of cyst formation by a novel tyrosine kinase inhibitor. Kidney Int
56: 406 – 413, 1999
46. Sommardahl CS, Woychik RP, Sweeney WE, Avner ED,
Wilkinson JE: Efficacy of taxol in the orpk mouse model of
polycystic kidney disease. Pediatr Nephrol 11: 728 –733, 1997
47. Cannon M, Barr F: Screening for polycystic kidney disease in
cats. Vet Rec 147: 639 – 640, 2000