© 2008 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
Gain-of-function mutations in TRPV4 cause autosomal
dominant brachyolmia
Matthew J Rock1, Jean Prenen2, Vincent A Funari1, Tara L Funari1, Barry Merriman3, Stanley F Nelson3,4,
Ralph S Lachman1,4,5, William R Wilcox1,4, Soraya Reyno6, Roberto Quadrelli7, Alicia Vaglio7,
Grzegorz Owsianik2, Annelies Janssens2, Thomas Voets2, Shiro Ikegawa8, Toshiro Nagai9,
David L Rimoin1,3,4,10, Bernd Nilius2 & Daniel H Cohn1,3,4
The brachyolmias constitute a clinically and genetically
heterogeneous group of skeletal dysplasias characterized by a
short trunk, scoliosis and mild short stature1. Here, we identify
a locus for an autosomal dominant form of brachyolmia on
chromosome 12q24.1–12q24.2. Among the genes in the
genetic interval, we selected TRPV4, which encodes a calcium
permeable cation channel of the transient receptor potential
(TRP) vanilloid family, as a candidate gene because of its
cartilage-selective gene expression pattern. In two families with
the phenotype, we identified point mutations in TRPV4 that
encoded R616Q and V620I substitutions, respectively. Patch
clamp studies of transfected HEK cells showed that both
mutations resulted in a dramatic gain of function characterized
by increased constitutive activity and elevated channel
activation by either mechano-stimulation or agonist stimulation
by arachidonic acid or the TRPV4-specific agonist 4a-phorbol
12,13-didecanoate (4aPDD). This study thus defines a
previously unknown mechanism, activation of a calciumpermeable TRP ion channel, in skeletal dysplasia pathogenesis.
Endochondral ossification requires the orchestrated actions of regulatory and structural molecules that together are responsible for linear
bone growth. Early in development, regulatory factors, including
transcription factors, receptors and their ligands, pattern the early
axial and appendicular skeletal anlage (reviewed in ref. 2). Subsequently, the cartilage model ossifies and the growth plates of endochondral bones are established. Proliferation and expansion of growth
plate chondrocytes followed by apoptosis and mineralization require
additional regulatory factors, including the PTHrP/PTHR and
FGF/FGFR3 regulatory pathways as well as structural components of
the cartilage extracellular matrix. Frequently, quantitative or qualitative defects in these molecules lead to skeletal dysplasia phenotypes3.
Indeed, many of the diverse molecules that are essential for normal
skeletal development have been identified by the characterization of
genes associated with skeletal dysplasias.
The brachyolmias are a heterogeneous group of skeletal dysplasias
of unknown etiology that primarily affect the spine. At least three
types of brachyolmia have been described1. Type 1 brachyolmia
includes the Hobaek and Toledo forms and is inherited in an
autosomal recessive fashion4,5. Both forms of type 1 brachyolmia are
characterized by scoliosis, platyspondyly with rectangular and elongated vertebral bodies, overfaced pedicles and irregular, narrow intervertebral spaces. The Toledo form is distinguished by corneal opacities
and precocious calcification of the costal cartilage. Type 2 brachyolmia
(Maroteaux type) is also an autosomal recessive disorder, primarily
distinguished from type 1 by rounded vertebral bodies and less
overfaced pedicles1,6. Some cases are associated with precocious
calcification of the falx cerebri. Type 3 brachyolmia is an autosomal
dominant form with severe kyphoscoliosis and flattened, irregular
cervical vertebrae7. Paradoxically, although the limbs are mildly
shortened in all three types of brachyolmia, they show minimal
epiphyseal and metaphyseal abnormalities on radiographs.
Figure 1 shows the pedigree of a large family (International Skeletal
Dysplasia Registry (ISDR) reference number R99-102) with autosomal
dominant brachyolmia. The clinical phenotype was characterized
by moderately short-trunk short stature, mildly short limbs, mild
brachydactyly and no extraskeletal clinical findings. The most characteristic radiographic features were scoliosis with platyspondyly and
overfaced pedicles, which were most prominent in the lumbar
vertebrae (Fig. 2a,b). There were mild irregularities at the metaphyses
of the proximal femora (Fig. 2c) and the hands showed delayed
epiphyseal and carpal ossification (Fig. 2d).
Using a two-stage genome scan (see Methods), we identified a locus
for the phenotype at chromosome 12q24.1–24.2 with a maximum lod
1Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA. 2Katholieke Universiteit (KU) Leuven, Department of Molecular Cell
Biology, Laboratory for Ion Channel Research, Campus Gasthuisberg, B-3000 Leuven, Belgium. 3Departments of Human Genetics, 4Pediatrics and 5Radiological
Sciences, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California, USA. 6Servicio de Traumatologı́a y Ortopedia,
Hospital Pereira Rossel, Montevideo, Uruguay. 7Instituto de Genética Médica, Hospital Italiano, Montevideo, Uruguay. 8Laboratory for Bone and Joint Diseases, SNP
Research Center, RIKEN, Tokyo, Japan. 9Department of Pediatrics, Koshigaya Hospital, Dokkyo University, Saitama, Japan. 10Department of Medicine, David Geffen
School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, California, USA. Correspondence should be addressed to D.H.C. ([email protected]).
Received 20 November 2007; accepted 23 April 2008; published online 29 June 2008; doi:10.1038/ng.166
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p.R616Q, in the fifth transmembrane region
of TRPV4 (ref. 9). This change was not
present among 107 alleles of ancestry1
2
3
4
5
8-10 11-12
matched unaffected individuals. Similarly, all
6*
7 *
3
2
II
five affected individuals studied in family
193 201
R99-457 carried a 858G4A transition that
266 266
97 99
228 244
predicted a V620I substitution. Supplemen256 260
174 176
tary Figure 2 online shows a diagram of
163 165
290 293
144 156
TRPV4 indicating the locations of the substitutions. A comparison of amino acid
1*
6
7
6-10
11
2*
3*
4*
5*
sequences among TRPV4 orthologs showed
5
III
that both Arg616 and Val620 are conserved
D12S78
193 191
179 195
179 187
266 268 266 266
D12S1613 270 266
266 268
97 99 107 107
D12S353 93 99
107 99
among the human, rat, mouse, chicken, stick242 240 242 226
D12S1583 240 244
242 230
260 260 264 256
D12S1646 260 262
264 264
leback and zebrafish proteins (SupplemenD12S79
167 167
161 169
161 165
163 163 163 163
D12S1718 165 163
163 163
293 301 293 293
D12S2082 293 293
293 297
tary Fig. 2 and ref. 9), suggesting that the
D12S86
159 165
138 146
138 134
sequence changes are likely to be pathogenic.
2*
3*
4*
7*
8*
1*
5*
6
10
11
9
IV
In addition, for Arg616, sequence alignment
191 179 179 189
185 179
197 197 191 179 191 195
of TRPV4 with the paralogous TRPV1 and
266 266 266 266
268 266
270 266 266 266 266 268
99 107 107 103
99 107
99 99
99 109 99 107
244 242 230 230
240 242
230 230 244 242 246 230
TRPV2 and the more highly selective calcium
262 264 264 264
260 264
256 256 262 264 262 264
167 161 174 172
167 161
163 167 167 161 167 169
163 163 163 163
163 163
163 163 165 163 163 163
channel proteins TRPV5 and TRPV6 showed
293 293 301 297
301 293
293 293 293 293 293 297
165 146 138 149
138 138
148 132 159 138 165 146
complete conservation (Supplementary Fig.
2*
3*
4*
5*
9
1*
6
7
8
2). Subsequently, sequence analysis of TRPV4
V
197 191
197 179
179 189 191 189
excluded structural gene mutations in two
266 266
266 266
266 266 266 266
109 99
109 107
107 103 99 103
additional autosomal dominant and two
230 244
230 242
244 230 244 230
256 262
256 264
262 264 262 264
167 167
167 161
167 172 167 172
sporadic brachyolmia cases, suggesting that
163 165
163 163
163 163 163 163
293 293
293 293
293 297 293 301
autosomal dominant brachyolmia may be
132 159
132 138
165 149 165 149
genetically heterogeneous.
Figure 1 Pedigree and haplotypes for family R99-102. Filled symbols, affected individuals; open
The TRPV4 channel (reviewed in refs. 10–
symbols, unaffected individuals; gray boxes, probable affected individuals; *, DNA sample collected.
13) gates in response to a large variety of
Microsatellite markers are listed to the left of generation III. The haplotype segregating with the disease
stimuli, including cell swelling, warm temis boxed, and the shaded boxed loci were excluded by recombination mapping.
peratures, 4aPDD and endogenous lipid arachidonic acid. Activation by cell swelling and
score of 3.04 at a recombination fraction of zero for the marker at arachidonic acid requires cytochrome P450 (CYP) epoxygenase activity
locus D12S79. Two-point lod scores for five marker loci from this to convert arachidonic acid to epoxyeicosatrienoic acids, which then act
region are listed in Supplementary Table 1 online. We constructed as TRPV4 agonists11–14. The fraction of constitutively active channels
haplotypes according to the NCBI physical map (Fig. 1) and identified can be increased by elevation of intracellular Ca2+ or by heat10,14.
recombinations that limited the genetic interval transmitted with
To determine the effect of the brachyolmia-associated mutations on
the disorder.
TRPV4 activity, we expressed human TRPV4 in HEK cells. In comparBecause the brachyolmia phenotype is restricted to the skeleton, we ison to wild-type TRPV4 (Fig. 4a,b), the R616Q channel yielded a
hypothesized that the gene involved would be selectively expressed in
cartilage. We assessed microarray data from cartilage and noncartilage
tissues8 to define the expression profiles of genes within the 11.1-Mb
interval defined by recombination mapping. Expression analysis for all
probe sets within the interval showed that TRPV4 had about tenfold
higher cartilage-selectivity than the average of all other genes (Fig. 3).
On the basis of these data, we analyzed the TRPV4 coding region
and exon–intron boundaries for mutations in the index family and
in a second family (R99-457) with a similar autosomal dominant
brachyolmia phenotype.
We found heterozygosity for single-base changes within exon 12 of
TRPV4 in both families (Supplementary Fig. 1 online). In family R99102, a 1847G4A transition was found exclusively among affected
family members and predicted a single amino acid substitution,
a
b
1
2
© 2008 Nature Publishing Group http://www.nature.com/naturegenetics
I
Figure 2 Radiographs of the proband at age 8 years, 3 months. (a) Anteriorposterior view of the spine showing scoliosis and overfaced pedicles.
(b) Lateral view of the spine showing anterior rounding of the vertebrae
accompanied by flattening of the vertebral bodies. (c) Anterior-posterior
view of the proximal femur and acetabulum showing mild irregularity at
the proximal femoral metaphyses. (d) Anterior-posterior view of the hands
showing delayed ossification.
1000
c
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function’ phenotype by significantly increasing the fraction of constitutively open chan25
nels and by potentiated agonist activation.
8
20
These results demonstrate that the muta6
15
tions lead to the brachyolmia phenotype
by increasing constitutive and stimulus4
10
dependent TRPV4 activity13. The increase in
2
5
constitutive open channel activity is similar
0
0
to that resulting from in vitro substitutions
D12S353
D12S86
including Y591A in the TRPV4 TM4
domain17, F707A in the sixth transmembrane
Figure 3 Gene expression analysis throughout the linked region of chromosome 12. Shown are 230
helix and E797A or E797K in the TRPV4 C
Affymetrix probe sets representing genes within 12q24.1–24.2 in chromosomal order across the
bottom. Data for TRPV4 are marked by the arrow. Cartilage-selective expression (defined in ref. 8) is
terminus18. The consequences of the Y591A
plotted using the right y axis (black line), and the level of cartilage expression compared to noncartilage
substitution and a second substitution
tissue (purple bars) or dedifferentiatied chondrocytes (yellow bars) is plotted on the left y axis. Median
(R594Q), which is similar to the brachyolmia
cartilage selectivity (computed from the linear regression of all points) is represented by the dashed
mutations, were suggested to reveal involveblack line. The boundary of the linked region is denoted by the marker loci shown on the x axis.
ment in the general gating mechanism of
TRPV4 (ref. 17). The data presented here
much larger constitutive current before agonist application (Fig. 4c,d). are thus compatible with the hypothesis that Arg616 and Val620 are
The shape of the IV curve and the reversal potentials were not changed structurally important residues that modulate gating function, causing
(Fig. 4b,d). Activation with the TRPV4-specific agonist 4aPDD also an increased Ca2+ influx both under resting and stimulated conditions.
resulted in significantly increased currents. The data show significantly
Recently, it has been shown that overexpression of mouse TRPV4 in
increased constitutively activated current in the mutant (Fig. 4e). zebrafish caused marked shortening and curvature of the axial
Similar data were obtained for the mutation encoding V620I, with a skeleton19. These developmental abnormalities might be caused by
significantly increased constitutive current at +100 mV, albeit some- uncontrolled accumulation and activation of TRPV4 at the cell surwhat less increased than for the R616Q mutant, but a markedly face, which in turn compromises normal calcium homeostasis. A
increased maximal 4aPDD-activated current (Supplementary Fig. 3 newly identified TRPV4 binding protein, OS-9, diminishes this effect
online). Cell surface biotinylation assays showed no significant differ- by reducing the TRPV4 traffic to the plasma membrane. Thus,
ences in the level of TRPV4 at the plasma membrane between the wild overexpression of normal TRPV4 may have similar consequences as
type and mutants (Supplementary Fig. 4 online), excluding the the activating mutations found in individuals with brachyolmia.
possibility that the increased channel activity was due to altered channel Notably, knockout of Trpv4 in the mouse resulted in mice of normal
trafficking. In addition, mechano-activation by hypotonic cell swelling size and growth parameters20.
(reduction of extracellular osmolarity from 320 to 200 mOsm) and
The data presented here contribute to increasing evidence that
channel activation by application of 10 mM arachidonic acid15,16 TRPV4 is a key regulatory molecule in the growth plate21. Although
markedly increased the activity of both mutant channels (Fig. 5). the specific mechanism by which TRPV4 activation causes brachyolThus both the R616Q and V620I substitutions confer a ‘gain-of- mia is unknown, there are several possibilities. First, pharmacological
12
30
a
b
hTRPV4
3,000
c
I (pA/pF)
R616Q
d
+4αPDD
3,000
1,500
1,500
100
–1,500
–100 mV
+100 mV
–3,000
Control
+4αPDD
500 pA/pF
V (mV)
–100
10 s
I (pA/pF)
+4αPDD
V (mV)
500 pA/pF
© 2008 Nature Publishing Group http://www.nature.com/naturegenetics
Selectivity
Relative expression
10
–1,500
10 s
Figure 4 Expression of human TRPV4 and the R616Q mutant in HEK293 cells. (a) Time course
obtained from voltage ramps measured at –100 mV (black boxes) and +100 mV (red circles). 1 mM
4aPDD was applied at the time indicated. HEK cells were transfected with human wild-type TRPV4.
The data reflect typical activation of TRPV4. Before application of the specific agonist 4aPDD,
currents measured at –100 mV and +100 mV reflect constitutively open channels. After application
of 4aPDD, more channels were activated, causing an increase in current at both potentials.
(b) Current voltage relationships obtained from the time course shown in a and measured at the
points indicated by the filled circles. Note that the IV curves reverse at slightly positive potentials and
show a modest inward and outward rectification as typical for TRPV4. No such currents were observed
in nontransfected or mock transfected cells (that is, those transfected with the GFP plasmid without
the TRPV4 construct (data not shown)). (c) Same experiment as in a; however, cells were transfected
with the R616Q mutant of TRPV4. Note the increased current at both potentials before activation
with 4aPDD. (d) IV curves from the experiment shown in c. (e) Summarized data from the
constitutive current and the 4aPDD activated current measured at –100 mV and +100 mV
from wild-type TRPV4 and the mutant channel (mean ± s.e.m., *P o 0.05, n ¼ 14).
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100
–100
–100 mV
+100 mV
e
Control
+4αPDD
–3,000
I (nA/pF)
2
WT
*
D616Q
+100 mV
*
1
0
–1,000
–100 mV
Control
(constitutive)
1 µM 4αPDD
1001
© 2008 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
Figure 5 Expression of TRPV4 in HEK 293 cells.
hTRPV4
R616Q
V620I
Same experiments as in Figure 4 and
3
2 I (nA/pF)
2 I (nA/pF)
2 I (nA/pF)
I (nA/pF)
V620I
Supplementary Figure 3, except that TRPV4 was
R616Q
Constitutive
2
+100 mV
1
stimulated by hypotonic cell swelling (200 mOsm
1
1
200 mOsm
1
hTRPV4
instead of 320 mOsm) and application of 10 mM
V (mV)
V (mV)
V (mV)
0
arachidonic acid (AA). (a) Current voltage
–100 mV
–100
100 –100
100
100 –100
–1
relationships (IV) for wild type, R616Q and
320 mOsm (constitutive)
–1
–1
–1
V620I upon activation by cell swelling. Note the
200 mOsm
unchanged shape of the IV curves obtained from
R616Q
V620I
hTRPV4
voltage ramps of the controls before the stimulus
V620I
3
I (nA/pF)
I (nA/pF)
1
2 I (nA/pF)
2 I (nA/pF)
(black traces represent the constitutive activity;
2
Constitutive
+ 100 mV
R616Q
red traces reflect activity during stimulation).
1
1
10 µM AA
1
(b) Current activation by 10 mM arachidonic acid
hTRPV4
V (mV)
V (mV)
V (mV)
0
measured from voltage ramps at –100 mV and
–100 mV
100 –1
–100
100 –100
100 –100
+100 mV. (c) Pooled data from the same series
–1
–1
Control (constitutive)
of experiments as shown in a for wild-type TRPV4
10 µM AA
(n ¼ 6), R616Q (n ¼ 7) and V620I (n ¼ 7).
Averaged values before stimulation (white bars, averaged and s.e.m.) and maximal values during stimulation by hypotonic cell swelling (gray bars). Note that
all data on mutants are significantly higher as compared with that of wild type (P o 0.05). Current values are shown for +100 mV and –100 mV. (d) Pooled
data from same series of experiments as shown in b for wild-type TRPV4 (n ¼ 6), R616Q (n ¼ 8) and V620I (n ¼ 8). Data are averaged values and s.e.m.
Note that values for mutants are significantly higher (P o 0.05) compared with that for wild type, for both constitutively active channels and after
stimulation with 200 mOsm or arachidonic acid.
a
c
b
d
activation of TRPV4 induces Sox9 expression in ATDC5 cells21. Sox9
has an essential role in chondrocyte differentiation in the growth
plate, and both increased and decreased Sox9 can cause defects in
endochondral ossification. Haploinsufficiency for Sox9 in humans
causes campomelic dysplasia22. In contrast, a mutation in Prkg2 that
results in the persistence of Sox9 in growth plate chondrocytes causes
growth retardation in the Komeda miniature rat Ishikawa23. Thus,
Sox9 is tightly titrated, and increased Sox9 due to TRPV4 activation
could affect growth plate chondrocyte differentiation.
Second, TRPV4 has a functional role in keratinocyte cell volume
regulation24. The tenfold volumetric increase in hypertrophic chondrocytes contributes up to 80% of longitudinal endochondral bone
growth25. Perhaps activation of TRPV4 in chondrocytes decreases the
extent of chondrocyte hypertrophy during endochondral ossification.
Finally, TRPP1 and TRPP2 (also known as PKD1 and PKD2) have
been localized in cilia-like structures of osteoblasts and osteocytes, and
PKD1-null mice develop articular cartilage and growth plate
defects26,27. TRPV4 associates with TRPP2 (ref. 28) and, together
with TRPV2, has been implicated in the regulation of cilia activity in
the bronchioles11. This suggests that TRPV4 activation could possibly
lead to altered ciliary function.
This study describes activating mutations in TRPV4 that result in an
autosomal dominant form of brachyolmia. The mutations increase
constitutive TRPV4 activity, and identify an essential role for cation
channel activity in the growth plate. Abnormal skeletogenesis resulting
from alterations in calcium homeostasis is a previously unknown
mechanism in the etiology of skeletal dysplasias, and the specific
skeletal abnormalities observed in brachyolmia reveal a particularly
important function for this process in the growth and stability of the
spine. The nature of the mutations also suggests that modulation of
TRPV4 activity using calcium channel inhibitors could represent an
avenue for treatment.
METHODS
Subjects. Under an IRB protocol approved by Cedars-Sinai, we collected blood
and isolated DNA from cases referred through the ISDR. Informed consent was
obtained for all family members. We determined the clinical status of each
family member by clinical and radiographic criteria.
Genome scan. We carried out an initial microsatellite genome scan using DNA
from 7 family members and 384 microsatellite markers from the ABI Prism
1002
Linkage Mapping Set Version 2 (Applied Biosystems). We identified seven
chromosomal regions with lod scores of 41.0, and we analyzed these with
additional microsatellite markers identified through the Genome Database using
DNA from 15 family members. Amplified PCR products were resolved by gel
electrophoresis on an ABI Prism 377 DNA sequencer and output genotypes
were processed using ABI Prism Genescan version 3.1.2 software for allele size
determination. We then imported genotypes into the linkage analysis program
Mendel 4.0 and carried out two-point linkage analysis to calculate lod scores29.
Brachyolmia was modeled as an autosomal dominant, fully penetrant disease
with an allele frequency of 0.0001. The allele frequencies for each marker were
set at 1/N, where N was the number of alleles observed in the pedigree.
Mutation detection. PCR fragments covering exons 2 to 16 of the TRPV4
coding sequence were amplified from 100 ng of genomic DNA using AmpliTAQ
polymerase and standard PCR protocols in the Gene Amp PCR System 9700
(Applied Biosystems). The primers used (at an annealing temperature
of 60 1C) are listed in Supplementary Table 2 online. Exon 1 was amplified
using the Qiagen HotStart Hifidelity kit and buffer Q according to the
manufacturer’s instructions, using a touchdown PCR reaction with the annealing temperature starting at 65 1C and decreasing 1 1C every 3 cycles until
reaching 62 1C, at which the reaction was cycled a further 25 times. Sequences of
amplified PCR products were determined using the ABI Prism Big-Dye
Terminator Cycle Sequencing Kit, version 3.1 (Applied Biosystems), and
analyzed on an ABI Prism 377 DNA Sequencer (Applied Biosystems). Sequence
data were processed using ABI software and analyzed using Sequencher
(Genecodes). We compared the DNA sequences against the cDNA reference
sequence for TRPV4, with nucleotide numbering starting from the A of the ATG
initiation codon of the reference sequence. Mutations and polymorphisms were
confirmed in two separate PCR products using bidirectional sequencing.
Allele frequency analysis. The sequence change detected in TRPV4 in family
R99-102 was within an Hpy188I restriction endonuclease cleavage site. The
change altered the recognition sequence from [TCC.GA]TTC to
[TCCAA]TTC, ablating the Hpy188I site in the putative mutant allele. The
target sequence was amplified with primers for exon 12 using DNA from more
than 50 normal Ashkenazi Jewish controls and cleaved with Hpy188I (New
England Biolabs). PCR products derived from an unaffected subject only
showed Hpy188I cleavage products of 180 bp and 117 bp. PCR products
derived from an affected individual showed one uncleaved PCR product of
297 bp in addition to the two cleavage products derived from the normal allele.
Expression of TRPV4 and the R616Q and V620I variants in HEK293 cells.
We grew human embryonic kidney cells HEK293 in DMEM containing 10%
(v/v) human serum, 2 mM L-glutamine, 2 U/ml penicillin and 2 mg/ml
streptomycin at 37 1C in a humidity-controlled incubator with 10% CO2.
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For electrophysiology, the HEK293 cells were transiently transfected with
the human TRPV4 vector and cloned as a BamHI fragment into the BcII-site
of the pCAGGS/IRES-GFP vector, which allows detection of transfected cells
based on their green fluorescence when illuminated at 475 nm. For transfection,
we used L-alanyl-L-glutamine (Merck Eurolab) and GeneJuice Transfection
Reagent (Novagen). Green fluorescence–negative cells from the same batch
were used as controls. We introduced mutations in TRPV4 using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The nucleotide sequences
of the mutants were verified by sequence analysis of the corresponding cDNAs.
AUTHOR CONTRIBUTIONS
M.J.R., J.P., T.V., B.N., S.F.N. and D.H.C. designed the experiments. M.J.R., J.P.,
G.O., A.J., B.M. and V.A.F. carried out the experiments. T.L.F., R.S.L., W.R.W.,
S.R., R.Q., A.V., S.I., T.N. and D.L.R. ascertained and diagnosed the subjects.
M.J.R., B.N. and D.H.C. wrote the manuscript.
Cell surface biotinylation and immunodetection. HEK293 cells were transfected with equal amounts of pCAGGS or IRES-GFP vectors carrying wild-type
or mutant TRPV4 proteins (similar transfection efficiency) and, after overnight
growth, they were washed twice with PBS and incubated with 1 mM EZ-link
Sulfo-NHS-SS-Biotin (Pierce) for 30 min at 4 1C. To quench any nonreacting
biotin reagents, we washed cells once with ice-cold 50 mM Tris (pH 8.0) and
then twice with PBS. After whole-cell protein extractions in PBS supplemented
with 1.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail (10 mg/ml
leupeptin and antipain, 2 mg/ml chymostatin and pepstatin), biotinylated
proteins were precipitated with immobilized streptavidin, using the manufacturer’s protocol (Pierce), and analyzed by SDS-PAGE and immunodetection
with antibodies to TRPV4 (ref. 15), pan-cadherin (Abcam) and b-actin (Sigma).
1. Shohat, M., Lachman, R., Gruber, H.E. & Rimoin, D.L. Brachyolmia: radiographic and
genetic evidence of heterogeneity. Am. J. Med. Genet. 33, 209–219 (1989).
2. Kornak, U. & Mundlos, S. Genetic disorders of the skeleton: a developmental approach.
Am. J. Hum. Genet. 73, 447–474 (2003).
3. Superti-Furga, A., Bonafe, L. & Rimoin, D.L. Molecular-pathogenetic classification of
genetic disorders of the skeleton. Am. J. Med. Genet. 106, 282–293 (2001).
4. Horton, W.A., Langer, L.O., Collins, D.L. & Dwyer, C. Brachyolmia, recessive type
(Hobaek): a clinical, radiographic, and histochemical study. Am. J. Med. Genet. 16,
201–211 (1983).
5. Toledo, S.P.A. et al. Recessively inherited, late onset spondylar dysplasia and peripheral corneal opacity with anomalies in urinary mucopolysaccharides: a possible error
of chondroitin-6-sulfate synthesis. Am. J. Med. Genet. 2, 385–395 (1978).
6. Fontaine, G., Maroteaux, P., Farriaux, J.P. & Bosquet, M. Pure spondyloepiphyseal
dysplasia or brachyolmia. Arch. Fr. Pediatr. 32, 493 (1975).
7. Gardner, J. & Beighton, P. Brachyolmia: an autosomal dominant form. Am. J. Med.
Genet. 49, 308–312 (1994).
8. Funari, V.A. et al. Cartilage-selective genes identified in genome-scale analysis
of non-cartilage and cartilage gene expression. BMC Genomics 8, 165–177 (2007).
9. Liedtke, W. et al. Vanilloid receptor–related osmotically activated channel (VR-OAC),
a candidate vertebrate osmoreceptor. Cell 103, 525–535 (2000).
10. Nilius, B., Watanabe, H. & Vriens, J. The TRPV4 channel: structure-function relationship and promiscuous gating behaviour. Pflugers Arch. 446, 298–303 (2003).
11. Nilius, B., Owsianik, G., Voets, T. & Peters, J.A. Transient receptor potential cation
channels in disease. Physiol. Rev. 87, 165–217 (2007).
12. Pedersen, S.F., Owsianik, G. & Nilius, B. TRP channels: an overview. Cell Calcium 38,
233–252 (2005).
13. Nilius, B., Vriens, J., Prenen, J., Droogmans, G. & Voets, T. TRPV4 calcium entry
channel: a paradigm for gating diversity. Am. J. Physiol. Cell Physiol. 286,
C195–C205 (2004).
14. Watanabe, H. et al. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to
activate TRPV4 channels. Nature 424, 434–438 (2003).
15. Watanabe, H. et al. Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol
derivatives. J. Biol. Chem. 277, 13569–13577 (2002).
16. D’hoedt, D. et al. Stimulus-specific modulation of the cation channel TRPV4 by
PACSIN 3. J. Biol. Chem. 283, 6272–6280 (2008).
17. Vriens, J., Owsianik, G., Janssens, A., Voets, T. & Nilius, B. Determinants of 4 alphaphorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4.
J. Biol. Chem. 282, 12796–12803 (2007).
18. Watanabe, H. et al. Modulation of TRPV4 gating by intra- and extracellular Ca2+. Cell
Calcium 33, 489–495 (2003).
19. Wang, Y. et al. OS-9 regulates the transit and polyubiquitination of TRPV4 in the
endoplasmic reticulum. J. Biol. Chem. 282, 36561–36570 (2007); published online
11 October 2007.
20. Suzuki, M., Mizuno, A., Kodaira, K. & Imai, M. Impaired pressure sensation in mice
lacking TRPV4. J. Biol. Chem. 278, 22664–22668 (2003).
21. Muramatsu, S. et al. Functional gene screening system identified TRPV4 as a regulator
of chondrogenic differentiation. J. Biol. Chem. 282, 32158–32167 (2007).
22. Foster, J.W. et al. Campomelic dysplasia and autosomal sex reversal caused by
mutations in an SRY-related gene. Nature 372, 525–530 (1994).
23. Chikuda, H. et al. Cyclic GMP-dependent protein kinase II is a molecular switch from
proliferation to hypertrophic differentiation of chondrocytes. Genes Dev. 18,
2418–2429 (2004).
24. Becker, D., Blasé, C., Bereiter-Hahn, J. & Jendrach, M. TRPV4 exhibits a functional
role in cell-volume regulation. J. Cell Sci. 118, 2435–2440 (2005).
25. Hunziker, E.B. Mechanism of longitudinal bone growth and its regulation
by growth plate chondrocytes. Microsc. Res. Tech. 28, 505–519 (1994).
26. Olsen, B.R. et al. Genetic and epigenetic determinants of skeletal morphogenesis –
role of cellular polarity and ciliary function in skeletal development and growth.
Oral Biosci. Med. 2, 57–65 (2005).
27. Xiao, Z. et al. Cilia-like structures and polycystin-1 in osteoblasts/osteocytes and
associated abnormalities in skeletogenesis and Runx2 expression. J. Biol. Chem. 281,
30884–30895 (2006).
28. Giamarchi, A. et al. The versatile nature of the calcium-permeable cation channel
TRPP2. EMBO Rep. 7, 787–793 (2006).
29. Lange, K. et al. Mendel version 4.0: a complete package for the exact genetic analysis
of discrete traits in pedigree and population data sets. Am. J. Hum. Genet. 69 (Suppl.),
A1886 (2001).
30. Nilius, B., Prenen, J., Wissenbach, U., Bodding, M. & Droogmans, G. Differential
activation of the volume-sensitive cation channel TRP12 (OTRPC4) and volumeregulated anion currents in HEK-293 cells. Pflügers Archiv. Eur. J. Phys. 443,
227–233 (2001).
Electrophysiology. We measured whole-cell membrane currents with an EPC-9
(HEKA Elektronik, sampling rate, 1 ms; 8-Pole Bessel filter 3 kHz) using
ruptured patches. Patch electrodes had a DC resistance between 2 and 4 MO
when filled with intracellular solution. An Ag-AgCl wire was used as a reference
electrode. We monitored capacitance and access resistance continuously.
Between 50% and 70% of the series resistance was electronically compensated
to minimize voltage errors. We applied a ramp protocol, consisting of a voltage
step from the holding potential of 0 mV to –100 mV followed by a 400 ms linear
ramp to +100 mV. This protocol was repeated every 2 s. Cell membrane
capacitance values were used to calculate current densities. For electrophysiological measurements, the standard extracellular solution contained 150 mM
NaCl, 6 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose and 10 mM
HEPES, buffered at pH 7.4 with NaOH. The pipette solution was composed of
20 mM CsCl, 100 mM Asp, 1 mM MgCl2, 10 mM HEPES, 4 mM Na2ATP,
10 mM BAPTA and 2.93 mM CaCl2. The free Ca2+ concentration of this
solution is 50 nM. The osmolality of this solution, measured using a vaporpressure osmometer (Wescor 5500, Schlag), was 320 ± 5 mOsm. The non–
PKC-activating phorbol ester, 4a-phorbol 12,13-didecanoate (4aPDD, Alexis
Biochemicals), was applied at a 1 mM concentration from a 10 mM stock
solution in ethanol. Arachidonic acid was purchased from Sigma (applied from
a 10 mM DMSO stock to a final concentration of 10 mM). For hypotonic cell
swelling, we superfused cells with a solution containing 95 mM NaCl, 6 mM
CsCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose and 10 mM HEPES,
buffered to pH 7.4 with NaOH. Isoosmolarity was achieved by adding mannitol
(125 mM) to this solution to reach 320 mOsm. Hypotonic cell swelling
was induced by removing mannitol to lower the extracellular osmolarity to
200 mOsm (see ref. 30 for details). All measurements were carried out at room
temperature, 22–25 1C.
Statistics. We tested significance between individual groups using the unpaired
Student’s t-test (P o 0.05). Data are expressed as the mean ± s.e.m.
Accession codes. GenBank: human TRPV4 cDNA, NM_021625; human
TRPV4, NP_671737.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Institutes of Health
(HD22657) and the Human Frontiers Science Program (HFSP Research Grant
Ref. RGP 32/2004), the Belgian Federal Government, the Flemish Government, the
Onderzoeksraad KU Leuven (GOA 2004/07, F.W.O. G.0136.00; F.W.O. G.0172.03,
Interuniversity Poles of Attraction Program, Prime Ministers Office IUAP Nr.3P4/
23, Excellentiefinanciering EF/95/010) to B.N. Microarray data were generated and
analyses were performed within the University of California, Los Angeles DNA
microarray facility. We thank the families for their active participation.
NATURE GENETICS VOLUME 40
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NUMBER 8
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AUGUST 2008
Published online at http://www.nature.com/naturegenetics/
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