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Objective: Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. Design: DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. Results: The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. Conclusions: Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development.
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A dominant TRPV4 variant underlies osteochondrodysplasia in
Scottish fold cats
B. Gandoly
**
, S. Alamri z, W.G. Darby x, B. Adhikari k, J.C. Lattimer y, R. Malik ,
C.M. Wade #, L.A. Lyons y, J. Cheng k, J.F. Bateman yy, P. McIntyre x, S.R. Lamand
ez
a
,
B. Haase #
*a
yCollege of Veterinary Medicine, University of Missouri, Columbia, MO, USA
zMurdoch Childrens Research Institute, and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Australia
xSchool of Medical Sciences, RMIT University, Bundoora, Australia
kComputer Science Department, Informatics Institute, C. Bond Life Science Center, University of Missouri, Columbia, MO, USA
Centre for Veterinary Education, University of Sydney, Sydney, Australia
#Faculty of Veterinary Science, University of Sydney, Sydney, Australia
yy Murdoch Childrens Research Institute and Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Australia
article info
Article history:
Received 15 December 2015
Accepted 25 March 2016
Keywords:
TRPV4
Feline
Bone
Cartilage
Cat
Variant
summary
Objective: Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteo-
chondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and pro-
gressive joint destruction. This study aimed to identify the gene and the underlying variant responsible
for the osteochondrodysplasia.
Design: DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array
and a caseecontrol genome-wide association analysis conducted. The gene encoding a calcium
permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4)was
identied as a candidate within the associated region and sequenced. Stably transfected HEK293 cells
were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to
activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2
stimulation.
Results: The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associ-
ated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats.
Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell
surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic
agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to
hypotonic conditions.
Conclusions: Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further
functional studies to identify how and why the mutations affect cartilage and bone development.
©2016 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
*Address correspondence and reprint requests to: B. Haase, Faculty of Veterinary Science, University of Sydney, Regimental Drive, B19-305 RMC Gunn, Sydney, 2006 NSW,
Australia. Tel: 61-2-8627-0277; Fax: 61-29351-3957.
** Address correspondence and reprint requests to: B. Gandol, College of Veterinary Medicine, Department of Veterinary Medicine and Surgery, Vet Med Building, 1600
East Rollins Street, University of Missouri, Columbia, MO, 65201, USA.
E-mail addresses: gandolb@missouri.edu (B. Gandol), sultan.alamri@mcri.edu.au (S. Alamri), bill.darby@rmit.edu.au (W.G. Darby), bap54@mail.missouri.edu
(B. Adhikari), lattimerJ@missouri.edu (J.C. Lattimer), richard.malik@sydney.edu.au (R. Malik), claire.wade@sydney.edu.au (C.M. Wade), lyonsla@missouri.edu (L.A. Lyons),
chengji@missouri.edu (J. Cheng), john.bateman@mcri.edu.au (J.F. Bateman), peter.mcintyre@rmit.edu.au (P. McIntyre), shireen.lamande@mcri.edu.au (S.R. Lamand
e),
bianca.waud@sydney.edu.au (B. Haase).
a
Joint senior authors.
http://dx.doi.org/10.1016/j.joca.2016.03.019
1063-4584/©2016 The Authors. Published by Elsevier Ltd and Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Osteoarthritis and Cartilage xxx (2016) 1e10
Please cite this article in press as: GandolB, et al., A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats,
Osteoarthritis and Cartilage (2016), http://dx.doi.org/10.1016/j.joca.2016.03.019
Introduction
Osteochondrodysplasias (or skeletal dysplasias) are a hetero-
geneous group of disorders caused by structural, metabolic and
endocrine defects that compromise cartilage and/or bone growth
and give rise to a malformed skeleton
1e3
. Skeletal development is a
highly complex process inuenced by a vast number of genes and
precise tuning of these genes is essential for normal skeletal
development. Although individual skeletal dysplasias are rare in
humans, research into their pathogenesis has provided valuable
insights into bone and cartilage development, as well as more
common connective tissue disorders such as osteoarthritis
2,4e6
.
One of the biggest challenges in the post-genome era is ana-
lysing and dissecting complex and quantitative traits. While such
analyses remain a challenge, companion animals provide powerful
model populations to study underlying genetic mechanisms. As
most dog and cat breeds have been developed within the last 200
years, the remarkable phenotypic and genetic diversity observed
today is the result of intense articial selection. Founder effects at
breed creation and subsequent line-breeding have resulted in the
unique genetic background of breeds with extensive linkage
disequilibrium and long haplotype blocks, compared to human
populations
7,8
. While this process has reduced the overall genetic
heterogeneity within breeds, it has also enriched alleles with large
effects on favourable traits. This population structure is highly ad-
vantageous for genome-wide association studies (GWAS) as fewer
markers and fewer individuals are required to map even complex
traits
9
. An undesirable consequence of the intensive selection for
breed-specic traits has been the accumulation of various disease
alleles and a high frequency of genetic disorders in many dog and
cat breeds. For a remarkable number of these, analogous human
genetic disorders are known (http://omia.angis.org.au/home/).
Recent advances in feline genomics, including a whole-genome
reference sequence
10
, and a feline DNA genotyping array
11
,have
enabled GWAS in cats and allow the cat to be used as a model
species to gain insights into the molecular mechanisms underlying
human genetic disorders.
Scottish fold cats are characterized by their unique ear shape,
caused presumably by reduced resilience of the pinna cartilage.
While affected kittens are born with straight ears, the pinnae begin
to fold forward at around three to four weeks of age. Based on
pedigree information and breeding experiments, the Scottish fold
phenotype is inherited as a highly penetrant autosomal dominant
trait
12e14
. A congenital degenerative osteochondrodysplasia char-
acterised by malformations in the distal forelimbs, distal hind limbs
and tail was initially thought to be restricted to homozygous mutant
cats, but was subsequently identied in heterozygous mutant
cats
12,15,16
. Based on the initial nding the breeding recommenda-
tion for Scottish fold cats is to mate fold-eared cats only with normal
eared-cats to avoid producing homozygousmutant cats. Preliminary
histologic examinations suggested chondrocyte cell death in artic-
ular cartilage, and disturbed maturation of proliferative chon-
drocytes to hypertrophic chondrocytes in the growth plate
16
.
Radiographic examinations suggest defective endochondral ossi-
cation resulting in variably reduced length and abnormal shape of
the metatarsal and metacarpal bones, accompanied by accelerated
degenerative joint disease and progressive peri-articular new bone
formation. Interestingly, the age at onset of clinical signs, as well as
severity and the progression of the secondary new bone formation is
highly variable among affected heterozygous cats. Whether genetic
or environmental factors are responsible for the observed pheno-
typic differences is yet to be determined.
The present study used genetic, computational and in vitro
strategies to identify the gene underlying the Scottish fold osteo-
chondrodysplasia phenotype. A missense variant in the Transient
Receptor Potential Vanilloid family member 4gene (TRPV4) is asso-
ciated with ear folding, abnormal distal appendicular bones and a
progressive degenerative joint disorder with different degrees of
severity.
Material and methods
Animals and SNP array genotyping
All samples were collected with the permission of the cat's
owner, under University of California eDavis institution animal
care and use protocol 15933 and with approval of the University of
Sydney Animal Ethics Committee (N00/10-2012/3/5837). Cats were
sourced from different Scottish fold/Scottish shorthair populations
in Europe, Australia and the USA. Radiographs were available from
three severely affected Scottish fold cats. One severely affected cat
was studied using standard radiography, a helical computerised
tomography (CT) and scintigraphy following an intravenous
technetium-99m injection to highlight regions with increased bone
remodelling. EDTA anti-coagulated blood or non-invasive buccal
swab samples were collected from 44 cases (Scottish fold) and 54
controls (22 Scottish shorthairs estrait eared siblings of cats with
folded ears, 14 British shorthairs, 13 Selkirk rex and ve Persian
cats). Genomic DNA was isolated using the DNAeasy Kit (Qiagen,
Hilden, Germany) and concentrated using the Genomic DNA Clean
&Concentrator Kit (Zymo Research, Irvine, USA) when necessary.
Individual genotypes were determined using the Illumina Innium
Feline 63K iSelect DNA array.
Genome-wide association study and ne mapping
Array marker locations were adjusted to the most recent feline
genome assembly Felis catus 6.2/felCat5
11
. A caseecontrol genome-
wide association analysis was performed as previously described
11
.
The population substructure within cases and controls was evalu-
ated using a multi-dimensional scaling (MDS) plot with two di-
mensions and the identical-by-state (IBS) allele-sharing proportion
was calculated for each individual using PLINK
17
. Outlier samples
and individuals with a proportion of IBS >0.3 were excluded. The
GWAS results were adjusted for multiple testing (-mperm 100,000)
using PLINK
17
. The threshold for genome-wide signicance was
Bonferroni-adjusted. Linkage disequilibrium and haplotypes in the
region from position 23,500,000 to 25,5000,000 of feline chro-
mosome D3 were determined using HAPLOVIEW
18
. The corre-
sponding syntenic region in the mouse genome was searched for
potential candidate genes using the Mouse Genome Browser
(http://gbrowse.informatics.jax.org/cgi-bin/gb2/gbrowse/
mousebuild38/).
PCR and variant analysis
Five cats including two cases (Scottish folds) and three straight-
eared controls (one Scottish-shorthair and two domestic short-
hairs) were selected for initial variant detection. Primers to amplify
the 15 TRPV4 coding exons (exons 2e16) including the 5
0
- and 3
0
-
untranslated regions were designed using Primer 3 software
(http://biotools.umassmed.edu/bioapps/primer3_www.cgi).
Primer sequences are listed in Table S1. PCR products were ampli-
ed using the SequalPrep long range PCR kit (Invitrogen, Carlsbad,
USA) according to the manufacturer's protocol. An additional
primer set was designed for exon 6. PCR products for exon 6 were
amplied in 20
m
l reactions using AmpliTaq Gold according to the
manufacturer's protocol (Applied Biosystems, Foster City, USA). PCR
products were treated with shrimp alkaline phosphatase (Roche,
Basel, Switzerland) and exonuclease I (New England Biolabs,
B. Gandolet al. / Osteoarthritis and Cartilage xxx (2016) 1e102
Please cite this article in press as: GandolB, et al., A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats,
Osteoarthritis and Cartilage (2016), http://dx.doi.org/10.1016/j.joca.2016.03.019
Ipswich, USA), then directly sequenced using the PCR primers as
well as internal sequencing primers when necessary. Sequencing
products were separated using an ABI 3730 capillary sequencer
(Applied Biosystems, Foster City, USA), analysed using Sequencher
4.8 (GeneCodes, Ann Arbor, USA) and compared to the feline
reference sequence felCat5 September 2011 build. The candidate
causative variant was genotyped in all additional Scottish fold and
Scottish shorthair samples available (42 Scottish fold and 21 Scot-
tish shorthair). The possible impact of candidate variants on the
protein structure was investigated using PolyPhen-2 (http://
genetics.bwh.harvard.edu/pph2/).
Population screening
An allele-specic PCR assay was developed to detect the
c.1024G>T polymorphism. Cats (n¼728) from 40 different pop-
ulations, including pure-breed and random bred cats, were ana-
lysed. Each 25
m
l PCR reaction contained template DNA, 1
m
Mof
each primer (forward FAM-AACTACCTGACAGAGAACCCG, the wild-
type reverse tgatCAGGTCGTACATCTTGGTGgC and the affected
reverse CAGGTCGTACATCTTGGcGAA), 1 PCR Buffer (Denville
Scientic Inc., South Plaineld, USA), 2.5 mM MgCl
2
, 1.0 mM each
dNTP, 0.02
m
l DMSO and 1.0 U Choice-Taq DNA polymerase (Den-
ville Scientic, Inc., South Plaineld, USA). Template specicity was
increased by incorporating a sequence mismatch in both reverse
primers (lower case letter) and the addition of four mismatched
bases (lowercase underlined) to create a visually detectable pro-
duce size difference. PCR amplicons were visualized on an ABI 3730
DNA Analyzer (Applied Biosystems, Foster City, USA) and analyzed
using STRand software
19
.
Feline TRPV4 homology modelling
The Protein Data Bank (PDB)
20
was searched for structural
templates for the feline TRPV4 protein (XP_003994899.1) using
PSI-BLAST
21
. The top ranked human template was used to construct
the 3D homology model. The feline wild-type sequence was aligned
to the human template using CLUSTAL-W
22
and the 3D homology
model of the aligned TRPV4 N-terminal domain (residues 148-397)
was illustrated using MODELLER
23
.
Expression of normal and mutant TRPV4 in HEK293 cells
Human TRPV4 cDNA in pcDNA5/FRT/TO (Invitrogen, Carlsbad,
USA) has been described previously
24
. Human and cat TRPV4 pro-
teins are the same length and the amino acid sequences are 97%
identical. The V342F mutation was introduced using the Quik-
Change
®
II XL Site-Directed Mutagenesis Kit (Agilent Technologies)
and the entire coding region of the cDNA construct was sequenced
to conrm that PCR errors had not been introduced. Flp-InT-
Rex-293 cells (Life Technologies, Carlsbad, USA) were transfected
using Lipofectamine(Life Technologies, Carlsbad, USA) and stably
transfected cells selected for ve passages with 5
m
g/ml blasticidin
and 100
m
g/ml hygromycin. TRPV4 expression was induced with
1
m
g/ml tetracycline for four hours.
Preparation of total cell protein and cell surface biotinylated
proteins and immunoblotting
Total cell protein and biotinylated cell surface proteins were
isolated and analyzed by immunoblotting as described previously
24
using a rabbit polyclonal TRPV4 antibody (ab39260, Abcam, Cam-
bridge, UK), a monoclonal anti-actin antibody (A3853, Sigma
Aldrich, St. Louise, USA), or an anti-transferrin receptor antibody
(13-6800, Life Technologies, Carlsbad, USA) and Alexa Fluor
®
-
conjugated secondary antibodies (Life Technologies, Carlsbad, USA).
Immunoblots were imaged using a TyphoonTRIO Variable Mode
Imager (GE Healthcare, Little Chalfont, UK) and bands quantied
using ImageQuant TL Software (GE Healthcare, Little Chalfont, UK).
Intracellular calcium measurement
Intracellular calcium ([Ca
2þ
]
i
) was measuredby uorescence with
a FlexStation III (Molecular Devices, Sunnyvale, USA), as described
previously
24
. A total of 16,000 cells/well were plated into poly-
L
-
lysine coated 384 well plates, grown for 48 h, TRPV4 expression
induced with tetracycline for 4 h, before cells were loaded with 2
m
M
Fura-2 AM (Molecular Probes, Eugene, USA) for 1 h in the presence of
0.01% pluronic F-127. Loading and experiments were done as
described before
24
. Emission intensity was measured at 520 nm in
response to the synthetic TRPV4 agonist GSK1016709A, hypo-
osmolarity (240 mOsm) and the protease-activated receptor 2
(PAR2)activating peptideSLIGRL (50
m
M; Sigma) for 120s, at intervals
of 5.86 s, using excitation wavelengths of 340 and 380 nm. Four in-
dependent FlexStation experiments, each with six determinations at
each point, were combined and expressed as means ±
S
.
E
.
M
(n¼4).
Curve tting and statistics were done using GraphPad Prism 6. One
way ANOVA was used for multiple comparisons when there was a
single treatment and two-way ANOVA for multiple comparisons
when there were multiple treatments or time points.
Results
Radiographic ndings
The skeletal phenotype in the Scottish fold cats heterozygous for
the missense mutation in exon 6 ranged from mild to severe. Ra-
diographs showed all metatarsal and metacarpal bones are short-
ened and malformed; some are bent with enlarged proximal and
distal epiphyses (Fig. 1,Fig. S1). Intertarsal and tarsometatarsal joint
spaces are irregular, indistinct and narrowed (Fig. S1). The corre-
sponding joints in the forelimbs are similarly affected, although
generally to a lesser extent. All Scottish fold cats showed peri-
articular new bone formation (Fig. 2,Fig. S2), which was along
the proximal metatarsal bones, and the distal tarsal bones. The
peri-articular new bone appeared smooth in plain radiographs, but
the superior resolution of CT and elimination of superimposition
demonstrated that new bone was actually laid down irregularly
(Fig. 2). The proximal limb joints, long bones of the limbs and the
vertebral column were unaffected.
Genome-wide association study
Ninety-eight cats (44 cases and 54 controls) were genotyped on
the Illumina Innium iSelect Feline 63K DNA genotyping array.After
quality check 46,203 SNPs, 35 Scottish fold and 32 controls (17
Scottish Shorthair, 8 Selkirk Rex, 3 British Shorthair and 4 Persian)
remained for analysis (Fig. S3 (A)e(B)). The analysis identied a
signicant association on feline chromosome D3, with one SNP at
position D3:24,861,228 bp reaching genome-wide signicance after
correction for multiple testing (P
raw
¼4.7$10
11
,P
genome
¼0.00001)
(Fig. 3). A haplotype block dened in Haploview
18
spanning the in-
terval from D3:24,380,099 bp to D3:25,202,262 bp, harbouring the
highest associated SNP, was identied with a frequency of 50% in
affected cats (Fig. S4). The haplotype was unique to cases, suggesting
association with the dominant trait. Based on the feline genome
assembly F. catus 6.2/felCat5, this haplotype block contained 23
annotated genes (Table S2). Investigation of the syntenic region in
the mouse genome identied TRPV4 as the strongest positional
candidate gene.
B. Gandolet al. / Osteoarthritis and Cartilage xxx (2016) 1e10 3
Please cite this article in press as: GandolB, et al., A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats,
Osteoarthritis and Cartilage (2016), http://dx.doi.org/10.1016/j.joca.2016.03.019
TRPV4 variant analysis
The TRPV4 coding sequence (GenBank accession no.
XM_003994850.2) was analysed in two cases and three controls.
Comparison of all 15 coding exons, including partial intronic
sequence, revealed 26 sequence variants (Table I). Eleven variants
were located in the coding sequence and ve changed the amino
acid sequence. Only one exonic variant showed concordance with
the folded ear phenotype. Both Scottish fold cats were heterozy-
gous for a missense variant located in exon 6 (c.1024G>T) that was
absent in three straight-eared cats. Direct sequencing of 63 cats
determined that all 21 Scottish shorthair cats were homozygous for
the wild-type allele, two Scottish fold cats were homozygous for
the variant allele and 39 Scottish fold cats were heterozygous. A
single Scottish fold cat tested homozygous wild-type.
Population screening
Screening of 648 cats representing several breeds and domestic
shorthair cats of unknown ear type demonstrated the c.1024G>T
substitution was detected only in the Scottish fold breed and was
absent in all other populations (Table S3).
Protein domain analysis
The variant leads to a non-conserved p.V342F amino acid sub-
stitution and was predicted highly damaging by PolyPhen-2 (score
0.98). Using PSI-BLAST search the human TRPV4 ankyrin repeat
domain (PDB code: 4dx1) and the brown rat TRPV1 ion channel
protein (PDB code: 3j5p), which both include the cat variant site,
were the top matches. Only three residues are different from
Fig. 1. Radiographs of a severely affected Scottish fold cat. (A) Dorsopalmar view of the distal forelimbs. Carpal and carpometacarpal joint spaces are narrowed. Metacarpal bones
are shortened. (B) Dorsomedial-plantarolateral oblique view of the distal hind limbs. Metatarsa l bones are shortened. A mass of smoothly marginated ankylosing new bone is visible
on the plantar aspect of the tibiotarsal, intertarsal and tarsometatarsal joints. Distal intertarsal and tarsometatarsal joint spaces are narrowed.
Fig. 2. Radiological ndings in the right distal hindlimb of a severely affected Scottish Fold cat. (A) Lateral radiograph. (B) Three-dimensional volume-rendered computed to-
mography image. (C) Bone phase scintigraphic image showing the irregularly proliferative area of active bone remodelling (arrows) associated with individual metatarsophalangeal
joints.
B. Gandolet al. / Osteoarthritis and Cartilage xxx (2016) 1e104
Please cite this article in press as: GandolB, et al., A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats,
Osteoarthritis and Cartilage (2016), http://dx.doi.org/10.1016/j.joca.2016.03.019
human in cat TRPV4 for residues 148 to 397 (Fig. S5). Fig. S5 illus-
trates the 3D structure of the cat TRPV4 protein region using human
TRPV4 as a template.
Functional analysis of TRPV4 wild-type and TRPV4 p.V342F in
HEK293 cells
To determine the functional consequences stably transfected
HEK293 Flp-In cells expressing either wild-type or mutant TRPV4
were produced. No TRPV4 was detected in untransfected HEK293
cells
24
; however, whole cell lysates from transfected cells contained
similar amounts of either wild-type or V342F mutant TRPV4
[Fig. 4(A)e(B)]. In HEK293 cells, TRPV4 has three wild-type iso-
forms; unglycosylated, a form substituted with a high mannose N-
linked oligosaccharide, and a complex N-glycosylated form
[Fig. 4(A)]. When compared to wild-type TRPV4, cells expressing
V342F mutant TRPV4 contained more of the high mannose form and
less of the complex glycosylated form. The localization of TRPV4 was
examined using cell surface biotinylation and streptavidin pull-
down, followed by immunoblotting to identify and quantitate cell
surface proteins. Compared to the wild-type TRPV4, mutant V342F
TRPV4 was poorly expressed at the cell surface [Fig. 4(C)e(D)],
indicating that the variant altered TRPV4 protein trafcking.
Intracellular calcium imaging was used to evaluate channel ac-
tivity. A small increase in the constitutive intracellular calcium
concentration ([Ca
2þ
]
i
) was detected in cells expressing wild-type
TRPV4 compared to non-transfected cells. Interestingly, [Ca
2þ
]
i
was much higher in cells expressing TRPV4 V342F [Fig. 5(A)],
indicating that the basal activity of the mutant channels is
increased. The response of the channels to the synthetic TRPV4
agonist GSK1016790A showed that intracellular calcium in non-
transfected cells was not changed by GSK1016790A [Fig. 5(B)]. By
contrast, cells expressing wild-type TRPV4 responded with a dose-
dependent increase in [Ca
2þ
]
i
. Cells expressing V342F mutant
TRPV4 also showed a GSK1016790A dose-dependent increase in
[Ca
2þ
]
i
, although the response was reduced compared to wild-type
expressing cells and the maximum [Ca
2þ
]
i
was signicantly lower
than in wild-type expressing cells [Fig. 5(B)].
As TRPV4 channels respond to changes in tonicity
25
, the
response of the wild-type and mutant channels to reduced osmo-
larity was studied [Fig. 5(C)]. Cells expressing wild-type TRPV4
responded to hypotonic conditions with a rapid increase in [Ca
2þ
]
i
,
Fig. 3. GWAS identied region associated with osteochondrodysplasia in Scottish fold cats. a. A Manhattan plot showing the negative log of the probability of association (P-value)
between individual markers and osteochondrodysplasia. Markers are shown in alternating colours representing different chromosomes. The upper plot shows P
raw
values and the
lower plot shows values after correction for multiple testing (P
genome
). Signicance of P1$10
8
is indicated with a dashed line, a dotted line represents association with P1$10
4
.
b. Haplotype unique to cases in the region of association, spanning from position 24,380,099bp to position 25,202,262bp of cat chromosome D3. The asterisk marks the variant
signicantly associated with osteochondrodysplasia. The haplotype region contains 24 genes (represented by grey lines), including TRPV4 (indicated in red).
B. Gandolet al. / Osteoarthritis and Cartilage xxx (2016) 1e10 5
Please cite this article in press as: GandolB, et al., A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats,
Osteoarthritis and Cartilage (2016), http://dx.doi.org/10.1016/j.joca.2016.03.019
followed by a sustained phase of increased [Ca
2þ
]
i.
The response of
V342F expressing cells to hypotonic conditions was similar; how-
ever, the peak response is greater and the increase in [Ca
2þ
]
i
during
the sustained phase is higher, even when the higher resting [Ca
2þ
]
i
is taken into account [Fig. 5(D)].
Another physiological TRPV4 activation mechanism, PAR2
stimulation of TRPV4 signalling, was investigated. Experimentally,
PAR2 can be activated by synthetic peptides that mimic the teth-
ered ligand and the response of HEK293 cells expressing wild-type
and V342F mutant TRPV4 to the PAR2 activating peptide SLIGRL
was tested. As previously reported, untransfected HEK293 cells
showed a rapid increase in [Ca
2þ
]
i
reecting release from intra-
cellular stores
26
. In cells expressing wild-type TRPV4 this was fol-
lowed by a sustained increase in [Ca
2þ
]
i
when compared to the
transient response seen in untransfected HEK293 cells [Fig. 5(E)].
TRPV4 V342F-expressing cells also had a sustained increase in
[Ca
2þ
]
i
in response to PAR2 activation, and [Ca
2þ
]
i
remained higher
than in wild-type expressing cells throughout. However, the
baseline adjusted increase in [Ca
2þ
]
i
was similar in wild-type and
mutant expressing cells indicating that the magnitude of the
response was similar [Fig. 5(F)].
Discussion
TRPV4 is expressed in a range of tissues, including chondrocytes,
osteoblasts and osteoclasts, where its correct activity is crucial for
cell differentiation and tissue homeostasis
5,27,28
. Accordingly, TRPV4
mutations are responsible for a spectrum of dominantly inherited
human skeletal dysplasias, including, autosomal dominant bra-
chyolmia (OMIM 113500), spondylometaphyseal dysplasia
Kozlowski type (OMIM 1842522), and metatropic dysplasia (OMIM
156530)
29e31
, as well as a premature arthropathy affecting hands
and feet, familial digital arthropathy brachydactyly (OMIM
606835)
24
. The TRPV4 skeletal dysplasias show remarkable
phenotypic variability and are sometimes accompanied by neuro-
degenerative disorders
2,24,32e36
. The skeletal dysplasia TRPV4 mu-
tations that have been examined functionally cause a gain of
function and there is evidence that the resulting change in channel
conductance determines the severity of the phenotype
37
. While gain
of function mutations are responsible for obvious human skeletal
malformations, the complete loss of functional TRPV4 has surpris-
ingly little effect on skeletal development in mice
5,28
.
The TRPV4 protein has six transmembrane domains and large N-
and C-terminalcytoplasmic domains.The N-terminal region contains
a single proline-rich domain and six nger-loop forming ankyrin
repeat domains
38,39
. The N- and C-terminal cytoplasmic domains
play an important role in forming the functional tetrameric cation
channel, in addition to anchoring the protein tothe cytoskeleton and
permitting interactions with other proteins
39e42
. The dominantly
inheritedfolded ear and osteochondrodysplasia in Scottish fold cats is
associated with a p.V342F substitution (c.1024G>T) in the fth
ankyrin repeat within the N-terminal cytoplasmic domain (Fig. S5).
While the same amino acid substitution has been reported in a hu-
man patient with metatropic dysplasia, the mutation has not been
functionally characterized
43
.Puried recombinant ankyrin repeat
domains containing the V342F mutation have reduced thermal sta-
bility compared to wild-type domains and signicantly weakened
ATP binding even though V342 has no direct interaction with ATP
38
.
In wild-type TRPV4, ATP binding leads to a more compact structure
made possible by exibility in nger loop 3 in the ankyrin repeat
region which stabilizes the N-terminal domain
38
. ATP sensitises
TRPV4 channel activity and so the combination of reduced thermal
stability, and altered ATP binding can begin to explain why the
p.V342F mutation is pathogenic
44
.
To overcome the calcium toxicity that results from extended
TRPV4 overexpression, a system was used where TRPV4 is only
expressed after induction with tetracycline. This system allows
TRPV4 trafcking to the cell surface to be readily compared. TRPV4
Table I
Sequence variants identied in the cat TRPV4 gene
Position (genomic DNA)*Sequence change (cDNA)yLocation Protein Scottish fold 1 Scottish fold 2 Scottish shorthair DSH 1zDSH 2
g.25129900 c.25 T>C Exon 2 Y9H T/C T/C T/C C/C T/T
g.25129903 c.28G>A Exon 2 A10T G/A G/A G/A G/G G/G
g.25129908 c.33A>G Exon 2 Silent G/G A/G A/G A/A A/A
g.25129911 c.63T>C Exon 2 Silent C/C T/C T/C T/T T/T
g.25129944 c.69C>T Exon 2 Silent C/C C/C C/T 0 C/C
g.25130127 c.252C>T Exon 2 Silent C/T C/C C/C C/C C/C
g.25132506 c. 387-69C>T Intron 2 C/T C/T C/T C/C C/C
g.25132759 c.559þ12A>G Intron 3 G/A G/A G/A G/G A/A
g.25136691 c.712þ31A>G Intron 4 A/G G/G G/G A/A A/A
g.25136847 c.712þ166C>G Intron 4 G/C G/C C/C G/G 0
g.25136866 c.712þ185C>T Intron 4 T/C T/C C/C T/T 0
g.25137266 c.713-152G>A Intron 4 A/G A/G A/G A/A A/A
g.25137280 c.713-138C>T Intron 4 C/T C/T C/T C/C C/C
g.25138284 c.854-87T>C Intron 5 C/T C/T C/T C/C 0
g.25138288 c.854-83T>C Intron 5 C/T C/T C/T C/C 0
g.25138480 c.963A>C Exon 6 Silent A/C A/C A/C C/C A/A
g.25138541 c.1024G>T Exon 6 V342F G/T G/T G/G G/G G/G
g.25138621 c.1104T>C Exon 6 Silent T/C T/C T/C T/T T/T
g.25140888 c.1153-20C>T Intron 6 C/T C/C C/C C/C C/C
g.25140902 c. 1153-6G>T Intron 6 G/T G/G G/G G/G G/G
g.25142301 c. 1333-17T>C Intron 7 C/T C/T C/C T/T T/T
g.25143778 c.1659-66T>C Intron 10 T/T T/C T/T C/C 0
g.25143830 c. 1659-14C>T Intron 10 C/C C/C C/T C/C C/C
g.25144020 c.1824þ10C>T Intron 11 T/C C/C C/C T/T T/T
g.25146975 c.2041G>A Exon 13 G681S G/G G/A G/G G/G G/G
g.25149751 c.2387G>A Exon 15 S796N G/A G/A G/A G/G G/G
*
Numbering refers to chromosome D3 accession ID GCA_000181335.2.
y
Numbering refers to accession number XM_003994850.1.
z
Domestic shorthair. Variations that change the amino acid sequence are highlighted in grey. The heterozygous change leading to a p.V342F amino acid substitution in
Scottish fold cats is bolded.
B. Gandolet al. / Osteoarthritis and Cartilage xxx (2016) 1e106
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forms a tetramer within the ER and is then trafcked to the cell
surface
45
. Two lines of evidence indicate that the V342F mutation
impairs channel assembly and trafcking. In whole cell lysates,
mutant TRPV4 has less complex N-linked glycosylation and
increased high mannose glycosylation, indicating delayed transit
from the ER to the Golgi. Secondly, reduced cell surface expression
of the mutant channels conrms that intracellular trafcking is
disturbed. Thus, the V342F variant likely changes the structure and
stability of the N-terminal region, protein interactions critical for
tetramer formation and/or stability are compromised and tetramer
formation is delayed.
Even though fewer mutant channels were present at the cell
surface, in unstimulated conditions the intracellular calcium con-
centration was higher in cells expressing mutant TRPV4 channels
than in wild-type expressing cells. This is consistent with electro-
physiology studies that show TRPV4 skeletal dysplasia mutations
increase the channel basal open probabilities
37
. Wild-type and
mutant channels responded differently to a range of activating
stimuli. The dose response of wild-type and mutant channels to the
synthetic agonist GSK1016790A was similar, indicating that the
drug was able to efciently activate both channels. Despite this, the
maximum response to GSK1016790A was lower in mutant
expressing cells. Since GSK1016790A is thought to drive TRPV4 to
its maximum activity
37
, the reduced maximum response likely re-
ects reduced cell surface expression of V342F mutant channels.
The V342F mutation did not impact the response of the channels to
PAR2 stimulation; however, a gain of function was seen in the
response of the mutant channels to hypotonic conditions. V342F
expressing cells showed a greater maximum response and had
higher sustained intracellular calcium than wild-type expressing
cells even after adjusting for the higher basal activity, indicating the
mutant channels are more sensitive to hypotonicity than wild-type
channels. By contrast, other TRPV4 skeletal dysplasia mutants,
D333G, R594H and A716S, showed either reduced or no activation
by hypotonic conditions in transiently transfected HEK293 cells
46
.
This could reect different experimental conditions but likely in-
dicates that the downstream pathogenic consequences of TRPV4
mutations are complex and their inuence on any individual
channel property is only partly informative. Together, these data
demonstrate that TRPV4 wild-type and V342F channels behave
very differently in response to a synthetic agonist and physiological
activation mechanisms, supporting the conclusion that the TRPV4
V342F substitution is pathogenic and causes the bone and joint
phenotype in Scottish fold cats.
Interestingly, the skeletal elements that are affected by the
TRPV4 V432F mutation are different in humans and cats. The child
with the V342F mutation was diagnosed with metatropic
dysplasia
43
. While only limited clinical information was reported,
the patient had a short trunk and kyphoscoliosis. A review of eleven
metatropic dysplasia patients from 20 weeks gestation to 70 years of
age found that progressive and severe kyphoscoliosis was a promi-
nent feature
47
. Other common features included platyspondyly,
Fig. 4. Wild-type and p.V342F mutant TRPV4 expression in stably transfected HEK293 cells. (A) Immunodetection of TRPV4 and
b
-actin (loading control) in whole cell lysates. Three
TRPV4 isoforms were detected; an unglycosylated form (1), a form substituted with a high mannose N-linked oligosaccharide (2), and a form where the high mannose oligo-
saccharide had been modied to a complex glycan (3). (B) Quantitating all three TRPV4 bands showed that wild-type and V342F mutant TRPV4 were expressed at similar levels
(P¼0.57). (C) Cell surface proteins were biotinylated then isolated using streptavidin beads and TRPV4,
b
-actin and transferrin receptor (loading control) detected by immuno-
blotting. The absence of a
b
-actin signal conrms minimal contamination with intracellular proteins. (D) Quantitation of the TRPV4/transferrin receptor ratio shows that V342F
mutant TRPV4 is poorly expressed at the cell surface compared to wild-type TRPV4 (P¼0.0006). Each point in (B) and (D) is a determination from an individual well. De-
terminations of multiple wells in two independent experiments are shown. Graphs show mean ±S.E.M. Data sets were compared with the Student's ttest.
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aring metaphyseal long bones and short stature. The hands and
feet had generalised brachydactyly but no major deformities or
abnormal bone growth. By contrast, the distal fore and hindlimbs
are severely affected in Scottish fold cats, while the long bones and
the spine are relatively spared. The effects of TRPV4 variants on
different skeletal elements could reect regional species specic
differences in biomechanical properties. The biomechanical forces
on the spine in a bipedal human are different to those in a quad-
ruped cat
48
, and similarly, forces through the feet in a cat that both
jumps up and lands from heights will be greater than in most
humans. TRPV4 is a mechanosensor in chondrocytes
49
. Our data
shows V342F mutant TRPV4 has an elevated response to hypoto-
nicity, a surrogate measure of mechanical stress, compared to wild-
type TRPV4 and this altered transduction of mechanical signals
likely inuences chondrocyte intracellular responses and ultimately,
the disease presentation.
Dissecting the complex downstream consequences of TRPV4
skeletal dysplasia variants will require the appropriate cell types e
Fig. 5. Intracellular calcium levels in stably transfected HEK293 cells. (A) Constitutive internal uorescence ratio (**P<0.01, ****P<0.0001, one way ANOVA). (B) Changes in
intracellular calcium concentration in response to the synthetic TRPV4 agonist GSK1016790A. Cells expressing wild-type TRPV4 showed a dose dependent increase in intracellular
calcium that reached a plateau at higher concentrations. Cells expressing V342F mutant TRPV4 had higher intracellular calcium at low drug doses and reduced responses to
GSK1016790A at 10
7.0.
and 10
6.5
M(*P<0.05, two way ANOVA). (C) Changes in intracellular calcium concentration in response to hypotonicity (240mOsm). The response is
characterised by a rapid rise in intracellular calcium followed by a period of lower but sustained increase in intracellular calcium over basal levels. (D) To compare the magnitude of
the hypotonicity responses the constitutive uorescence ratio at time 0 (unstimulated) was subtracted from all other time points. Cells expressing V342F mutant TRPV4 had a higher
peak response and maintained a higher response than wild-type expressing cells over the course of the experiment (*P<0.05, two way ANOVA). (E) Changes in intracellular calcium
in response to PAR2 activating peptide (PAR2-AP). There is a rapid increase in intracellular calcium reecting release from internal stores followed by a period of sustained elevated
intracellular calcium in cells expressing wild-type and mutant TRPV4. (F) To compare the magnitude of the PAR2 responses the unstimulated uorescence ratio at time 0 was
subtracted from all other time points. When compared in this way wild-type and V342F mutant TRPV4 responded in a similar manner to PAR2 activation although the peak
response was higher in mutant expressing cells than wild-type expressing cells (*P<0.05, two way ANOVA). All graphs show mean ±S.E.M.
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chondrocytes, osteoblasts, and osteoclasts eexpressing wild-type
and mutant TRPV4 alleles, rather than widely used heterologous
cell expression systems. Chondrocytes from patients with TRPV4
variants are generally inaccessible and until now the only animal
models that have been produced are transgenic mice expressing
the Trpv4 V620I or R594H skeletal dysplasia mutations
50,51
under
the control of the Col2a1 promoter. While these mice had a rangeof
skeletal abnormalities similar to those found in human patients, the
TRPV4 R594H transgenic mice were generally more severely
affected than expected from the intermediate human spondylo-
metaphyseal dysplasia Kozlowski type that results from the R594H
mutation
50
. This failure to accurately reproduce the human
phenotype could stem from both inappropriately high TRPV4
expression in cartilage and inappropriate expression in the hyper-
trophic region of the growth plate cartilage. While the Col2a1
promoter is active in the hypertrophic region and the TRPV4
transgene product was expressed there
50,51
, TRPV4 is not normally
expressed in these terminally differentiated chondrocytes
24
. Access
to a naturally-occurring TRPV4 variant in a large animal model
opens the door to further functional studies concerning how and
why the mutation affects cartilage and bone development, and the
pathogenesis of accelerated osteoarthritis.
Author contribution
BG, SRL, JFB, LAL, PM and BH designed research. BG, SA, BD, BA,
JCL, SRL and BH performed research. LAL, RM JCL contributed case
material, radiographs and reagents. BG, SA, BD, CMW, PM, RM, SRL
and BH analysed data. SRL, BG and BH wrote the paper. All authors
read and approved the submitted manuscript version.
Competing interest statement
The authors have declared that they have no competing interests.
Role of the funding source
This study was supported by the Cat Health Network (D12FE-514),
the Morris Animal Foundation (D12FE-021), the National Health
and Medical Research Council of Australia (1025715 and 1043837),
the Victorian Government's Operational Infrastructure Support
Program, previously by the National Institutes of Health National
Center for Research Resources R24 RR016094 and is currently
supported by the Ofce of Research Infrastructure Programs/OD
R24OD010928 and the University of Missouri eColumbia
Gilbreath-McLorn Endowment (LAL). Richard Malik is supported by
the Valentine Charlton Bequest. Study sponsors played no role in
the study design, collection, analysis or interpretation of data.
Acknowledgements
The authors would like to thank all cat owners and breeders for
their support. Sarah Davies contributed to radiographic interpre-
tation and nomenclature.
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.joca.2016.03.019.
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... Individual breeds of companion and farm animals have historically been bred selectively for specific morphological and behavioral features. While there is huge phenotypic diversity seen across cat breeds, most breeds have been created in only the last 200 years (Gandolfi et al. 2016). Selection for extreme morphological features can lead to breed-specific health issues (Malik 2001). ...
... The distinctive phenotype of the Scottish Fold is caused by a dominant variant in the TRPV4 gene and results in a defective calcium-permeable, nonselective cation channel involved in multiple physiologic functions including aspects of normal cartilage (Gandolfi et al. 2016). This results in the ears folding forward and variable effects on articular cartilage and bone. ...
... Each of these studies report cases of severe SFOCD in cats younger than four years, which makes misdiagnosis of osteoarthritis unlikely. Gandolfi et al. (2016) reported genetic data for 44 Scottish Fold cats, including three severely affected heterozygous Scottish Fold cats for which radiographs were available, as well as one homozygous cat. Each of these cats showed classic radiological signs of SFOCD. ...
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The unique appearance of Scottish Fold cats is caused by a single gene variant in TRPV4, which impacts the development of cartilage. This results in the ears folding forward and variable effects on articular cartilage and bone. While some find this appearance desirable, early work demonstrated that homozygous cats with two copies of this variant develop severe radiographic consequences. Subsequent breeding programs have mated heterozygous cats with straight-eared cats to ensure an equal mix of heterozygous (fold) and wild-type (nonfolded) offspring, in the hope of raising healthy cats. More recent radiological surveys suggest that these heterozygous cats may also have medical problems consisting of deformed distal extremities in the worst cases and accelerated onset of osteoarthritis. However, these previous studies were undermined by selection biases, lack of controls, unblinded assessment and lack of known genotypes. Our aim was to determine if heterozygous cats exhibit radiological abnormalities when controlling for these limitations. Specifically, DNA and radiographs were acquired for 22 Scottish Fold cats. Four reviewers, blinded to the ear phenotype, assessed the lateral radiographs. Genotyping showed that all 10 folded-ear cats were heterozygous, and none of the straight-ear cats (n = 12) had the abnormal TRPV4 variant. Although each reviewer, on average, gave a numerically worse ‘severity score’ to folded-ear cats relative to straight-ear cats, the images in heterozygous cats showed much milder radiological signs than previously published. This study provides additional information to be considered in the complicated debate as to whether cats with the TRPV4 variant should be bred for folded ears given the potential comorbidities.
... Since the introduction of a feline genotyping array (Feline Illumina Infinium Array) which characterises ~ 63 000 variants across the feline genome, GWAS have become possible 45,46 . Published feline GWAS have focused on rare pedigree related conditions where clear phenotypic cases and controls are available [47][48][49][50][51] . Such studies have successfully identified monogenic mutations resulting in profound phenotypic change. ...
... congenital myasthenic syndrome in the Devon Rex 48 or hypokalemia in the Burmese 86 or for dominant traits that are under positive selection e.g. Scottish Fold cat folded ears 47 . However, exploration of complex traits is fundamentally vital to veterinary medicine, given that these represent the most common medical conditions that are impacting the health and welfare of the largest number of feline patients. ...
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Hypertension (HTN) and chronic kidney disease (CKD) are common in ageing cats. In humans, blood pressure (BP) and renal function are complex heritable traits. We performed the first feline genome-wide association study (GWAS) of quantitative traits systolic BP and creatinine and binary outcomes HTN and CKD, testing 1022 domestic cats with a discovery, replication and meta-analysis design. No variants reached experimental significance level in the discovery stage for any phenotype. Follow up of the top 9 variants for creatinine and 5 for systolic BP, one SNP reached experimental-wide significance for association with creatinine in the combined meta-analysis (chrD1.10258177; P = 1.34 × 10–6). Exploratory genetic risk score (GRS) analyses were performed. Within the discovery sample, GRS of top SNPs from the BP and creatinine GWAS show strong association with HTN and CKD but did not validate in independent replication samples. A GRS including SNPs corresponding to human CKD genes was not significant in an independent subset of cats. Gene-set enrichment and pathway-based analysis (GSEA) was performed for both quantitative phenotypes, with 30 enriched pathways with creatinine. Our results support the utility of GWASs and GSEA for genetic discovery of complex traits in cats, with the caveat of our findings requiring validation.
... The current 63 k Illumina feline single nucleotide polymorphism (SNP) mapping array has been used successfully to map variants for Mendelian diseases with breeds. Examples include the discovery of the WNK4 variant that causes hypokalemia in Burmese cats (8), a region on chromosome E1 associated with progressive retinal atrophy in Persian cats (9), a causal variant in COLQ for hereditary myopathy in Devon Rex and Sphynx cats (10), refinement of the region on chromosome B4 associated with craniofacial structure and frontonasal dysplasia in Burmese cats (11), a region on chromosome A3 associated with an inherited neurologic syndrome in a family of Oriental cats (12), and a dominant channelopathy variant causing osteochondrodysplasia in Scottish Fold cats (13). This array has also been used in a limited number of within-breed genome wide association studies (GWAS) for complex disease (14,15), but there are no reports of GWAS performed with an across-breed design. ...
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The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.
... For example, highly brachycephalic breeds such as the modern Persian and Exotic short hair can experience a range of health conditions associated with their eyes, skin, and respiration [143,144]. In the Scottish Fold, their characteristic 'folded' ears are the result of selection for heritable gene mutations, which also cause abnormal bone and cartilage development, leading to chronic pain and mobility issues [145]. The presence of such chronic conditions has the potential to induce poor mood, increased irritability, and human-and conspecific-directed aggression, in addition to a range of other behaviours which owners tend to find problematic [146][147][148]. ...
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Sociality can be broadly defined as the ability and tendency of individuals to reside in social groups with either conspecifics and/or other species. More specifically, sociability relates to the ability and tendency of individuals to display affiliative behaviours in such contexts. The domestic cat is one of the most globally popular companion animals and occupies a diverse range of lifestyles. Despite an arguably short period of domestication from an asocial progenitor, the domestic cat demonstrates an impressive capacity for both intra- and interspecific sociality and sociability. At the same time, however, large populations of domestic cats maintain various degrees of behavioural and reproductive autonomy and are capable of occupying solitary lifestyles away from humans and/or conspecifics. Within social groups, individuals can also vary in their tendency to engage in both affiliative and agonistic interactions, and this interindividual variation is present within free-living populations as well as those managed in confined environments by humans. Considerable scientific enquiry has focused on cats’ social behaviour towards humans (and conspecifics to a much lesser extent) in this latter context. Ontogeny and human selection, in addition to a range of proximate factors including social and environmental parameters and individual cat and human characteristics, have been highlighted as important moderators of cats’ sociability. Such factors may have important consequences regarding individuals’ adaptability to the diverse range of lifestyles that they may occupy. Where limitations to individuals’ social capacities do not enable sufficient e.g. adaption, compromises to their wellbeing may occur. This is most pertinent for cats managed by humans, given that the physical and social parameters of the cats’ environment are primarily dictated by people, but that positive human-selection for traits that enhance cats’ adaptability to such lifestyles appears to be limited. However, limitations in the availability and quality of evidence and equivocal findings may impede the current understanding of the role of certain factors in relation to cat sociability and associations with cat wellbeing, although such literature gaps also present important opportunities for further study. This review aims to summarise what is currently known about the various factors that may influence domestic cats’ sociality and sociability towards both humans and conspecifics, with a predominant focus on cats managed by humans in confined environments. Current limitations, knowledge gaps, and implications for cat wellbeing are also discussed.
... In cats, only a few skeletal dysplasias have been characterized at the molecular level. Osteochondrodysplasia in Scottish fold cats is caused by a missense variant in the TRPV4 gene (000319-9685) [6,7]. A form of chondrodysplasia that represents the breed standard in short-legged Munchkin cats is caused by a large deletion within the UGDH gene encoding UDP-glucose 6-dehydrogenase (OMIA 000187-9685) [8,9]. ...
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We investigated a highly inbred family of British Shorthair cats in which two offspring were affected by deteriorating paraparesis due to complex skeletal malformations. Radiographs of both affected kittens revealed vertebral deformations with marked stenosis of the vertebral canal from T11 to L3. Additionally, compression of the spinal cord, cerebellar herniation, coprostasis and hypogangliosis were found. The pedigree suggested monogenic autosomal recessive inheritance of the trait. We sequenced the genome of an affected kitten and compared the data to 62 control genomes. This search yielded 55 private protein-changing variants of which only one was located in a likely functional candidate gene, LTBP3, encoding latent transforming growth factor β binding protein 3. This variant, c.158delG or p.(Gly53Alafs*16), represents a 1 bp frameshift deletion predicted to truncate 95% of the open reading frame. LTBP3 is a known key regulator of transforming growth factor β (TGF-β) and is involved in bone morphogenesis and remodeling. Genotypes at the LTBP3:c.158delG variant perfectly co-segregated with the phenotype in the investigated family. The available experimental data together with current knowledge on LTBP3 variants and their functional impact in human patients and mice suggest LTBP3:c.158delG as a candidate causative variant for the observed skeletal malformations in British Shorthair cats. To the best of our knowledge, this study represents the first report of LTBP3-related complex skeletal dysplasia in domestic animals.
... 11 The exact cause of osteochondrodysplasia in Scottish Fold cats is unknown; however, the TRPV4 gene has been associated with this condition. 12 This condition follows an autosomal dominant inheritance with incomplete penetrance, and cats with homozygous fold-eared genes are severely affected by this disease, whereas cats with heterozygous genes tend to develop mild clinical signs. 13 Although our case did not receive mutational analysis, abnormality of the vertebrae may be associated with the osteochondrodysplasia seen in this breed. ...
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Case summary A 2-year-old neutered female Scottish Fold cat was presented with an 8-week history of progressive back pain, paraparesis and decrease of postural reactions in both pelvic limbs. MRI showed spinal cord compression from both ventral sides, which originated from the T4 vertebral body and pedicle. The lesion compressing the spinal cord had a bone-like density on CT, and endoscopic surgery was performed to excise it. Histopathological examination of the resected tissue showed no evidence of malignancy and the lesion was diagnosed as vertebral hypertrophy. After surgery, the neurological status of the cat gradually improved. The cat was ambulant at the follow-up evaluation 2 weeks after surgery. Six months later, hindlimb paresis had improved considerably, and no recurrence was observed on CT. Relevance and novel information This is the first description of thoracic vertebral canal stenosis due to hypertrophy of a single vertebra in a young cat. Excision of the hypertrophic vertebra by endoscopic surgery is less invasive than open surgery and may give a good prognosis.
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In the largest DNA-based study of domestic cats to date, 11,036 individuals (10,419 pedigreed cats and 617 non-pedigreed cats) were genotyped via commercial panel testing elucidating the distribution and frequency of known disease, blood type, and physical trait associated genetic variants across cat breeds. This study provides allele frequencies for many disease-associated variants for the first time and provides updates on previously reported information with evidence suggesting that DNA testing has been effectively used to reduce disease associated variants within certain pedigreed cat populations over time. We identified 13 disease-associated variants in 47 breeds or breed types in which the variant had not previously been documented, highlighting the relevance of comprehensive genetic screening across breeds. Three disease-associated variants were discovered in non-pedigreed cats only. To investigate the causality of nine disease-associated variants in cats of different breed backgrounds our veterinarians conducted owner interviews, reviewed clinical records, and invited cats to have follow-up clinical examinations. Additionally, genetic variants determining blood types A, B and AB, which are relevant clinically and in cat breeding, were genotyped. Appearance-associated genetic variation in all cats is also discussed. Lastly, genome-wide SNP heterozygosity levels were calculated to obtain a comparable measure of the genetic diversity in different cat breeds. This study represents the first comprehensive exploration of informative Mendelian variants in felines by screening over 10,000 pedigreed cats. The results qualitatively contribute to the understanding of feline variant heritage and genetic diversity and demonstrate the clinical utility and importance of such information in supporting breeding programs and the research community. The work also highlights the crucial commitment of pedigreed cat breeders and registries in supporting the establishment of large genomic databases, that when combined with phenotype information can advance scientific understanding and provide insights that can be applied to improve the health and welfare of cats.
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In the largest DNA-based study of domestic cat to date, 11,036 individuals (10,419 pedigreed cats from 91 breeds and breed types and 617 non-pedigreed cats) were genotyped via commercial panel testing, elucidating the distribution and frequency of known genetic variants associated with blood type, disease and physical traits across cat breeds. Blood group determining variants, which are relevant clinically and in cat breeding, were genotyped to assess the across breed distribution of blood types A, B and AB. Extensive panel testing identified 13 disease-associated variants in 48 breeds or breed types for which the variant had not previously been observed, strengthening the argument for panel testing across populations. The study also indicates that multiple breed clubs have effectively used DNA testing to reduce disease-associated genetic variants within certain pedigreed cat populations. Appearance-associated genetic variation in all cats is also discussed. Additionally, we combined genotypic data with phenotype information and clinical documentation, actively conducted owner and veterinarian interviews, and recruited cats for clinical examination to investigate the causality of a number of tested variants across different breed backgrounds. Lastly, genome-wide informative SNP heterozygosity levels were calculated to obtain a comparable measure of the genetic diversity in different cat breeds. This study represents the first comprehensive exploration of informative Mendelian variants in felines by screening over 10,000 domestic cats. The results qualitatively contribute to the understanding of feline variant heritage and genetic diversity and demonstrate the clinical utility and importance of such information in supporting breeding programs and the research community. The work also highlights the crucial commitment of pedigreed cat breeders and registries in supporting the establishment of large genomic databases that when combined with phenotype information can advance scientific understanding and provide insights that can be applied to improve the health and welfare of cats.
Preprint
Full-text available
The current feline genotyping array of 63k single nucleotide polymorphisms has proven its utility within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.
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Little is known about the genetic changes that distinguish domestic cat populations from their wild progenitors. Here we describe a high-quality domestic cat reference genome assembly and comparative inferences made with other cat breeds, wildcats, and other mammals. Based upon these comparisons, we identified positively selected genes enriched for genes involved in lipid metabolism that underpin adaptations to a hypercarnivorous diet. We also found positive selection signals within genes underlying sensory processes, especially those affecting vision and hearing in the carnivore lineage. We observed an evolutionary tradeoff between functional olfactory and vomeronasal receptor gene repertoires in the cat and dog genomes, with an expansion of the feline chemosensory system for detecting pheromones at the expense of odorant detection. Genomic regions harboring signatures of natural selection that distinguish domestic cats from their wild congeners are enriched in neural crest-related genes associated with behavior and reward in mouse models, as predicted by the domestication syndrome hypothesis. Our description of a previously unidentified allele for the gloving pigmentation pattern found in the Birman breed supports the hypothesis that cat breeds experienced strong selection on specific mutations drawn from random bred populations. Collectively, these findings provide insight into how the process of domestication altered the ancestral wildcat genome and build a resource for future disease mapping and phylogenomic studies across all members of the Felidae.
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Hereditary eye diseases of animals serve as excellent models of human ocular disorders and assist in the development of gene and drug therapies for inherited forms of blindness. Several primary hereditary eye conditions affecting various ocular tissues and having different rates of progression have been documented in domestic cats. Gene therapy for canine retinopathies has been successful, thus the cat could be a gene therapy candidate for other forms of retinal degenerations. The current study investigates a hereditary, autosomal recessive, retinal degeneration specific to Persian cats. A multi-generational pedigree segregating for this progressive retinal atrophy was genotyped using a 63 K SNP array and analyzed via genome-wide linkage and association methods. A multi-point parametric linkage analysis localized the blindness phenotype to a ~1.75 Mb region with significant LOD scores (Z ≈ 14, θ = 0.00) on cat chromosome E1. Genome-wide TDT, sib-TDT, and case–control analyses also consistently supported significant association within the same region on chromosome E1, which is homologous to human chromosome 17. Using haplotype analysis, a ~1.3 Mb region was identified as highly associated for progressive retinal atrophy in Persian cats. Several candidate genes within the region are reasonable candidates as a potential causative gene and should be considered for molecular analyses. Electronic supplementary material The online version of this article (doi:10.1007/s00335-014-9517-z) contains supplementary material, which is available to authorized users.
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Activating mutations in TRPV4 are known to cause a spectrum of skeletal dysplasias ranging from autosomal dominant brachyolmia to lethal metatropic dysplasia. To develop an animal model of these disorders, we created transgenic mice expressing either wild-type or mutant TRPV4. Mice transgenic for wild-type Trpv4 showed no morphological changes at embryonic day 16.5, but did have a delay in bone mineralization. Overexpression of a mutant TRPV4 caused a lethal skeletal dysplasia that phenocopied many abnormalities associated with metatropic dysplasia in humans, including dumbbell-shaped long bones, a small ribcage, abnormalities in the autopod, and abnormal ossification in the vertebrae. The difference in phenotype between embryos transgenic for wild-type or mutant Trpv4 demonstrates that an increased amount of wild-type protein can be tolerated and that an activating mutation of this protein is required to produce a skeletal dysplasia phenotype. © 2014 American Society for Bone and Mineral Research
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Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4V620I channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of theTRPV4V620I mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4T89I mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.-Leddy, H. A., McNulty, A. L., Lee, S. H., Rothfusz, N. E., Gloss, B., Kirby, M. L., Hutson, M. R., Cohn, D. H., Guilak, F., Liedtke, W. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations.
Chapter
The Protein Data Bank (PDB) is the single, freely available, global archive of structural data for biological macromolecules. It is maintained by the wwPDB consortium consisting of the Research Collaboratory for Structural Bioinformatics (RCSB PDB), the Protein Data Bank in Europe (PDBe), the PDB Japan (PDBj) and the BioMagResBank (BMRB). This chapter describes the organization of the wwPDB, the systems in place for data deposition, annotation and distribution, and a summary of the services provided by the wwPDB member sites. Keywords: Protein Data Bank
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Protease-activated receptor 2 (PAR2 ) is expressed on nociceptive neurons, and can sensitise transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not understood. We have previously shown that PAR2 causes receptor-operated activation of TRPV4, and that TRPV4 null mice have attenuated PAR2 -stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms leading to PAR2 -induced TRPV4 activation and pain. Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (SERCA; thapsigargin), Gαq (UBO-QIC), tyrosine kinases (Bafetinib and Dasatinib), or PI3 kinases (Wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice. In non-transfected HEK293 cells, PAR2 activation caused a transient increase in intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 caused a sustained phase of [Ca(2+) ]i increase, which was inhibited by HC067047, Bafetinib and Wortmannin; but not by thapsigargin, UBO-QIC, Dasatinib or LY294002. Bafetinib but not Dasatinib significantly inhibited PAR2 -induced mechanical hyperalgesia in vivo. This study supports a role for tyrosine kinases in PAR2 -mediated receptor-operated gating of TRPV4, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2 -induced activation of TRPV4 and subsequent mechanical hyperalgesia identifies Bafetinib (which is in development in oncology) as a potential novel analgesic therapy.