Content uploaded by Ivan Duran
Author content
All content in this area was uploaded by Ivan Duran on Aug 03, 2016
Content may be subject to copyright.
Content uploaded by Ivan Duran
Author content
All content in this area was uploaded by Ivan Duran on Aug 03, 2016
Content may be subject to copyright.
Content uploaded by Ivan Duran
Author content
All content in this area was uploaded by Ivan Duran on Aug 03, 2016
Content may be subject to copyright.
1
An inactivating mutation in intestinal cell kinase, ICK, impairs hedgehog signaling and causes short rib-
polydactyly syndrome
S. Paige Taylor
2¶
,
Michaela Kunova Bosakova
1¶
, Miroslav Varecha
1
, Lukas Balek
1
, Tomas Barta
4
, Lukas
Trantirek
5
, Iva Jelinkova
1
, Ivan Duran
6
, Iva Vesela
3
, Kimberly N. Forlenza
6
, Jorge H. Martin
6
, Ales
Hampl
4
, University of Washington Center for Mendelian Genomics
7
, Michael Bamshad
8,9,10
, Deborah
Nickerson
10
, Margie L. Jaworski
11
, Jieun Song
12
, Hyuk Wan Ko
12
, Daniel H. Cohn
6,13,14
, Deborah
Krakow
2,6,13,*
, Pavel Krejci
1,6,15
1
Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic
2
Department of Human Genetics, University of California Los Angeles, Los Angeles, California, USA,
90095
3
Institute of Experimental Biology, Masaryk University, 62500 Brno, Czech Republic
4
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, 62500 Brno,
Czech Republic
5
Central European Institute of Technology, Masaryk University, 62500 Brno, Czech Republic
6
Departments of Orthopaedic Surgery, Human Genetics, Obstetrics and Gynecology, and Orthopaedic
Institute for Children, University of California Los Angeles, Los Angeles, California, USA, 90095
7
University of Washington Center for Mendelian Genomics, University of Washington, Seattle,
Washington, USA, 98195
8
Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA, 98195,
USA
9
Division of Genetic Medicine, Seattle Children’s Hospital, Seattle, WA, USA 98105
10
Department of Genome Sciences, University of Washington, Seattle, Washington, USA 98195
11
Virginia Commonwealth University, Richmond, Virginia, USA 23298
© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
HMG Advance Access published July 27, 2016
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
2
12
College of Pharmacy, Dongguk University-Seoul, Goyang, 410-820, Korea
13
International Skeletal Dysplasia Registry, University of California Los Angeles, Los Angeles,
California, USA, 90095
14
Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los
Angeles, California, USA, 90095
15
International Clinical Research Center, St. Anne's University Hospital, 65691 Brno, Czech Republic
¶
equal contribution
*
author for correspondence: Deborah Krakow (e-mail: DKrakow@mednet.ucla.edu)
Corresponding Author
Deborah Krakow, MD
David Geffen School of Medicine
BSRB
615 Charles E. Young Drive S
Room 410
Los Angeles, CA 90095
DKrakow@mednet.ucla.edu
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
3
ABSTRACT
The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal
disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and
concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS,
revealing a role for cilia function in skeletal development. To identify additional SRPS genes and
discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a
cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell
Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity,
resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage
growth plate structure, defective Hedgehog and altered ERK signaling. These data identify ICK as an
SRPS-associated gene and reveal that abnormalities in signaling pathways contribute to defective
skeletogenesis.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
4
INTRODUCTION
Mammalian skeletal development is a carefully orchestrated and precisely-timed sequence of events that
includes embryonic limb bud initiation and outgrowth, mesenchymal specification and condensation, cartilage
differentiation and bone ossification, and finally, postnatal growth and maintenance (1). This complex process
is regulated by a variety of integrated molecular mechanisms, many of which have been revealed by identifying
the genes associated with skeletal dysplasia phenotypes. In many instances unappreciated components not
previously known to be important to skeletogenesis have been identified, providing valuable insights into the
underlying biology of skeletal development.
The short rib polydactyly syndromes (SRPS or short-rib thoracic syndromes, SRTD, MIM 208500) are a
group of autosomal recessive, perinatal-lethal disorders that present with profound effects on the skeleton that
include a long narrow thorax, shortened and hypoplastic long bones, and polydactyly. Other organs are also
frequently affected, including the brain, heart, kidneys, pancreas, intestines and genitalia. Based on clinical and
radiographic phenotypes, SRPS have historically been subdivided into types I through IV but as the associated
genes are identified, the nomenclature has continued to evolve (2). SRPS share genetic and phenotypic features
with other established skeletal ciliopathies, including asphyxiating thoracic dystrophy (Jeune syndrome), Ellis-
van Creveld dysplasia, and Sensenbrenner syndrome, consistent with the notion that these phenotypes constitute
a continuous spectrum of disease (3). Genetic studies demonstrating allelic heterogeneity among these disorders
support this concept.
SRPS are caused by mutations in genes involved in the function of primary cilia, microtubule-based
projections on the surface of nearly every cell that receive and integrate signaling inputs. Primary cilium
function depends on intraflagellar transport (IFT), the bi-directional transport system that shuttles ciliary
components, such as the tubulin building blocks, receptors, and signaling components, into and out of this
organelle. The IFT machinery is composed of two distinct complexes: the kinesin-2-driven anterograde IFT-B
complex, which moves components from the ciliary base to the tip, and the dynein-2-driven retrograde IFT-A
complex, which transports components from the tip to the base. Mutations in genes encoding IFT-A and IFT-B
components cause SRPS, including: IFT80 (MIM 611263) (4,5), WDR19 (MIM 614376) (6), WDR34 (MIM
615633) (7,8), WDR35 (MIM 614091) (9,10), IFT140 (MIM 266920) (11,12), IFT172 (MIM 615630) (13),
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
5
WDR60 (MIM 615462) (14), TTC21B (MIM 612014) (15), DYNC2H1 (MIM 603297) (16,17), DYNC2LI1(18),
CEP120 (19), KIAA0586 (20,21).
ICK is a MAP-like kinase belonging to the ancient yet poorly understood family of the tyrosine kinase
gene v-ros cross-hybridizing kinase (RCK) serine-threonine kinases. The RCK kinases, which include male
germ-cell associated kinase (MAK) and MAPK/MAK/MRK-overlapping kinase (MOK), are highly conserved
and have been implicated in cilia biology. ICK was first localized in the intestinal crypt, where it regulates
proliferation by promoting G1 cell cycle proliferation (22), and has since been shown to be ubiquitously
expressed. ICK is a substrate of cell cycle-related kinase (CCRK), a negative regulator of ciliogenesis and cilia
length (23), and ICK homologs in Caenorhabditis elegans (dyf-5) (24), Chlamydomonas reinhardtii (LF4p)
(25), and Leishmania mexicana (LmxMPK9) (26), are also negative regulators of cilia length (24). Loss-of-
function mutations in ICK, p.R272Q and p.G120C, cause endocrine-cerebro-osteodysplasia (ECO) (MIM
612651) an autosomal recessive, neonatal-lethal disorder similar to SRPS (27, 28). Two recently characterized
ICK-knockout mouse models recapitulate several phenotypic findings in ECO syndrome and show findings
similar to SRPS that include polydactyly, shortened long bones, and an underdeveloped skeleton (29, 30). In this
study, we identify and characterize a novel inactivating mutation in ICK that produces SRPS and disrupts the
architecture of the cartilage growth plate, alters hedgehog signaling, and further expands our understanding of
the underlying biology that produces SRPS.
RESULTS
Homozygosity for an inactivating mutation in ICK produces SRPS
To identify additional SRPS genes, exome sequencing was carried out under an approved human subjects
protocol in a cohort of SRPS cases. In one individual (International Skeletal Dysplasia Registry reference
number R05-024A, Fig. 1A) we identified homozygosity for a point mutation, c.238G>A (NM_014920),
predicting the amino acid substitution p.E80K in Intestinal Cell Kinase (ICK). The affected male was initially
identified by prenatal ultrasound with features of a severe skeletal dysplasia associated with multiple congenital
abnormalities, including hydrocephalus, genital abnormalities and craniofacial dysmorphism, and delivered at
38 weeks gestation. He was intubated for respiratory insufficiency but succumbed within a few hours after birth.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
6
Radiographs showed a long narrow thorax with short ribs, elongated clavicles, rhizomelia, mesomelia with
bending, a hypoplastic ileum and polydactyly (Fig. 1B-D, Table 1) findings consistent with SRPS.
Homozygosity for a different ICK missense mutation was characterized in the original case of ECO (27),
demonstrating that these are allelic phenotypes.
ICK residue E80 lies within the highly conserved serine/threonine kinase domain and forms a direct
bond with ATP as visualized by structural modeling (Fig. 2A, B). In silico substitution of the negatively
charged E80 for the positively charged K80 disrupts the electrostatic potential at the ICK-ATP interface, likely
compromising ATP-binding (Fig. 2B) and kinase activity. To test this prediction, we performed an in vitro cell-
free kinase assay using purified ICK
WT
or ICK
E80K
and myelin basic protein (MBP) as the substrate.
Phosphorylation of MBP was achieved with ICK
WT
but not with ICK
E80K
, indicating that the p.E80K substitution
abrogates kinase activity (Fig. 2C).
Having established that ICK
E80K
is kinase-dead, we next asked if this would prevent activation of the
protein. Activation of ICK is regulated by dual-phosphorylation of its T
157
-D-Y
159
motif: auto-phosphorylation
of Tyr
159
achieves basal activation and CCRK-mediated phosphorylation of Thr
157
achieves full activation (31).
Because Tyr
159
phosphorylation reflects basal ICK activity, it can be used as a surrogate to estimate the levels of
ICK activation (32). Figure 2D shows that ICK
E80K
was unable to auto-phosphorylate at Tyr
159
, while ICK
R272Q
found in ECO syndrome (27) retained approximately one third of Tyr
159
phosphorylation compared to ICK
WT
.
We conclude that p.E80K is a loss-of-function mutation differing from ICK
R272Q
ICK
E80K
demonstrates altered subcellular localization
Previous studies have established that ICK functions as both a cytoplasmic and nuclear kinase, suggesting that it
shuttles between these two compartments (27, 32, 33). Immunohistochemistry performed on HEK293T cells
revealed significant differences between ICK
WT
and ICK
E80K
localization (Fig. 3A). While ICK
WT
localized
evenly between the nucleus and cytosol, ICK
E80K
localized predominantly, but not exclusively, to the nucleus.
The ECO mutant ICK
R272Q
was almost exclusively cytosolic as described before
(27, 32) (Fig. 3B). Although
we do not know the reason for this difference, it is important to note that different residues are mutated, the
substituted amino acids have different charge consequences, and the ECO mutation only partially impaired ICK
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
7
activity when compared to the total loss of function mutation p.E80K (Fig. 2C, D). Interestingly, Arg
272
is a part
of the conserved
269
PKKRP
273
motif which serves as nuclear localization sequence (NLS) necessary for tagging
ICK for nuclear import (32).
ICK kinase activity regulates ciliogenesis and ciliary localization
To investigate ICK function during ciliogenesis, we expressed ICK
WT
, ICK
E80K
and ICK
R272Q
in NIH3T3 mouse
embryonic fibroblasts. Overexpression of ICK
WT
inhibited cilia formation in these cells, similar to recently
published data (29). By contrast, weaker overexpression of ICK
WT
inhibited ciliogenesis to a lesser degree,
suggesting that the level of ICK activity correlates inversely with ciliogenesis (Fig. 4A-C). While strong
overexpression of ICK
E80K
inhibited ciliogenesis, weaker overexpression of ICK
E80K
did not, suggesting that the
kinase activity of ICK is required to negatively regulate ciliogenesis. In line with that, cells weakly
overexpressing partially inactive ICK
R272Q
formed cilia with frequency between ICK
WT
and ICK
E80K
(Fig. 4A).
We also observed that cells expressing ICK
WT
had shorter cilia than controls, and that expression of ICK
E80K
produced longer cilia as compared with both the ICK
WT
- or ICK
R272Q
-transfected cells and the control (Fig. 4G).
The cilia in the ICK
E80K
mutant cells were longer, with greater variability in length, than in ICK
WT
cells,
suggesting that ICK kinase activity correlates inversely with cilia length (Fig. 4G). Concordant with these
findings, longer cilia were also observed in cultured cells lacking ICK due to RNAi knockdown or in cells from
the Ick knockout mouse (29). Localization within the cilia also differed between the ICK variants; ICK
E80K
and
ICK
R272Q
predominantly localized to the tip of the cilia compared with ICK
WT
, which localized almost
exclusively to the cilia base (Fig. 4D, E). Interestingly, only a minority of ICK
R272Q
-transfected cells localized
the transgenic ICK to cilia, which was in contrast with ICK
WT
and ICK
E80K
cells where majority of cilia were
positive for ICK (Fig. 4F). R272Q thus impairs ICK ability to localize to both the nucleus and cilia. Altogether,
the loss of kinase activity in ICK
E80K
altered its subcellular localization, ciliogenesis, cilia length, and
distribution within primary cilia.
Cilia from R05-024A fibroblasts are long and twisted
We further addressed the ciliary architecture in patient-derived fibroblasts. R05-024A fibroblasts produced
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
8
longer cilia, on average, compared to control fibroblasts (Fig. 5A-C) in agreement with the ICK expression
studies in NIH3T3 cells (Fig. 4G). Scanning electron microscopy analysis showed that cilia from R05-024A
fibroblasts appeared thin and twisted as compared control fibroblast cilia (Fig. 5D). The evidence obtained from
both R05-024A primary fibroblasts and ectopic expression studies in NIH3T3 cells demonstrated that kinase-
inactive ICK
E80K
produced elongated cilia in agreement with previous studies on ICK mutations (29, 34).
Impaired GLI3 processing and Hedgehog (Hh) signaling in R05-024A fibroblasts
To investigate whether the ICK mutation impaired Hh signaling, we stimulated fibroblasts from R05-024A and
controls with smoothened agonist (SAG) (35) and quantified the resulting amounts of GLI3 full-length
(GLI3FL) and repressor (GLI3R) forms as readout for GLI3 activity. In the absence of pathway stimulation,
GLI3FL was normally processed into GLI3R, as demonstrated in control fibroblasts (Fig. 6A). R05-024A
fibroblasts demonstrated a higher baseline ratio of GLI3FL to GLI3R amounts as compared with control
fibroblasts (Fig. 6A). With SAG stimulation, control fibroblasts demonstrated an increased GLI3FL to GLI3R
ratio, indicative of pathway activation, yet in R05-024A fibroblasts there were negligible changes in this ratio
upon SAG treatment. In R05-024A fibroblasts, there was markedly less GLI3R relative to GLI3FL and GLI3
activated processing was impaired, suggesting that mutant ICK altered GLI3 activity. To investigate this further,
we visualized ciliary localized GLI3 and found that endogenous GLI3 accumulated to a far greater extent in
primary cilia tips in R05-024A relative to control fibroblasts (Fig. 6B). In addition, GLI3 accumulation in R05-
024A cilia persisted longer after exposure to SAG when compared with control cells, which showed similar
GLI3 levels with no accumulation over time, consistent with impaired GLI3 processing due to the ICK
E80K
mutation (Fig. 6B). To see whether impaired GLI3 processing lead to altered expression of Hh target genes, we
analyzed levels of GLI1 and PTCH1 transcripts. Compared to control fibroblasts, the R05-024A cells showed
higher basal levels of GLI1 and PTCH1 expression (Fig. 6C), which corresponds to less abundant repressor
GLI3 (GLI3R) in these cells (Fig. 6A). Treatment with SAG resulted in significantly lower induction of GLI1
and PTCH1 expression in R05-024A, altogether demonstrating defective response to Hh signal (Fig. 6D).
Ick
-/-
mouse cartilage growth plates show abnormalities in architecture and impaired Hh signaling
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
9
Because of the profound effect of ICK mutations on skeletal development in humans and mice, we investigated
the localization of ICK in the distal femoral cartilage growth plate of P1 WT mice. Histologic analyses
performed with ICK antibody showed ubiquitous ICK expression, with particularly higher expression in the
proliferating and prehypertrophic zone chondrocytes, and relatively less expression in hypertrophic
chondrocytes (Supplementary Fig. S1). Minimal expression was seen in the perichondrium and primary
spongiosum (Fig. S1). Similar to previous findings in transfected cells, the intracellular distribution of ICK was
both cytoplasmic and nuclear (Fig. S1). Because cartilage growth plate from case R05-024A was not available,
we examined growth plate cartilages of the Ick
-/-
animals (29). Picrosirius red staining of the control and Ick
-/-
growth plates revealed marked disruption of growth plate architecture, with a shortened proliferative zone and
poor column formation in the hypertrophic zone, with fewer cells contributing to the hypertrophic chondrocyte
columns in Ick
-/-
compared to Ick
+/-
growth plates (Fig. 7A, B). In addition, proliferating and hypertrophic
chondrocytes were subjectively smaller with less cytoplasm and diminished extracellular matrix between cells,
leading to the appearance of increased cellularity throughout the growth plate (Fig. 7A, B). The primary
spongiosum also showed diminished numbers of discrete trabeculae (Fig. 7A, B) and, similar to the radiographs
in the affected neonate and the Ick
-/-
skeletal preparations (29), there was bending at the mid-diaphyseal portion
of the humeral bone (Fig. 7A), as well as other bones in the appendicular skeleton (data not shown). The
proliferative and hypertrophic zone alterations suggest defects in both cellular proliferation and chondrocyte
differentiation, and the hypercellularity may result from an effect on extracellular matrix synthesis due to loss of
Ick.
Ihh is a well-recognized morphogenic organizer of the growth plate cartilage, which is produced by a
narrow zone of early hyperthrophic chondrocytes and is critical for proper chondrocyte progression from
proliferating to hypertrophic stages (36). The in situ hybridization analysis of Ihh expression in the growth
plates of E18.5 Ick
-/-
embryos
showed a normal distribution when compared to
Ick
+/-
(Fig. 7C). This contrasted
Gli1 and Ptch1 expression, which was virtually absent in proliferating chondrocytes and adjacent perichondrium
in the Ick
-/-
growth plates, demonstrating defective responsive to Ihh within the growth plate (Fig. 7C).
Altered ERK MAP kinase activity in R05-024A fibroblasts and Ick
-/-
growth plate chondrocytes
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
10
Cartilage growth plate analyses showed a decreased number of proliferating cells and ICK was highly expressed
in the proliferative zone. This suggested that ICK could be involved in the regulation of chondrocyte
proliferation and differentiation. Because it has been well established that elevated ERK activation inhibits
chondrocyte proliferation, induces premature senescence and disturbs chondrocyte differentiation (37, 38), we
tested the hypothesis that ERK activity might be altered in R05-024A cells. We found increased basal activating
phosphorylation of ERK and other members of ERK signaling module such as C-RAF and MEK in R05-024A
fibroblasts compared with control cells (Fig. 8A). Chemical inhibition of ERK pathway in R05-024A fibroblasts
did not significantly shorten cilia, suggesting that increased ERK activity is not responsible for the long cilia
seen in R05-024A fibroblasts (Fig. 8B, C). We next evaluated whether a similar increase in ERK activation
occurs in the growth plates of Ick
-/-
mice. Femoral and tibial growth plates were harvested and analyzed for ERK
activating phosphorylation by western blot. In contrast with the R05-024A fibroblast data, we found less ERK
phosphorylation in both femurs and tibias isolated from Ick
-/-
mice, demonstrating different ERK regulation by
ICK in fibroblasts and growth plate chondrocytes (Fig. 8D).
DISCUSSION
Here we show that homozygosity for a kinase-inactivating mutation in ICK, p.E80K, causes short rib
polydactyly syndrome (SRPS). Functional characterization of the p.E80K mutation revealed total loss of kinase
activity, malformed and elongated primary cilia, abnormal growth plate morphology and impaired GLI3
processing coupled with defective expression of GLI1 and PTCH1. Homozygosity for a different ICK missense
mutation, p.R272Q, produced ECO syndrome, a distinct disorder delineated in the Old Order Amish population
(27). Shared phenotypic findings between the two disorders include hydrocephalus, fused eyelids, small
palpebral fissures, abnormally shaped and rotated ears, genitourinary abnormalities, shortened long bones,
bowing of bones in the mesomelic segment and polydactyly. Thus mutations in ICK can produce an allelic
spectrum of disease from ECO to SRPS.
Residue E80 of ICK occurs within a highly conserved domain that is required for ATP binding; we
show that the mutation of this residue abolished kinase activity, likely by destabilizing the ATP-binding pocket.
We demonstrated loss of kinase activity in ICK
E80K
; first, purified ICK
WT
, but not ICK
E80K
, was able to
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
11
phosphorylate MBP in an in vitro kinase assay; second, phosphorylation of Tyr159, the site of ICK auto-
phosphorylation that reflects basal activation, was detected in lysates from cells expressing ICK
WT
, limited down
to one third
in ICK
R272Q
, but was nearly absent in ICK
E80K
. These findings suggest that the extent of ICK kinase
activity influences skeletogenesis, distinguishing the phenotypic consequences between ECO and SRPS
mutations.
We also showed that the kinase activity of ICK is important for both cilia structure and ciliogenesis.
Both patient-derived primary fibroblasts and NIH3T3 cells expressing ICK
E80K
produced longer cilia than their
respective controls. Additionally, the cilia from patient-derived fibroblasts appeared gaunt and twisted, similar
to the structures of cilia from Kif7 mutants Kif7
L130P
and Kif7
-/-
(39), suggesting that ICK may be required for
ciliary structural integrity. Further, expression of ICK
inhibited ciliogenesis in NIH3T3 cells in a dose-
dependent manner. Notably, ICK
WT
was a much stronger inhibitor of ciliogenesis than ICK
R272Q
, and weakly
expressed kinase-dead ICK
E80K
did not inhibit ciliogenesis at all, suggesting that the regulation of ciliogenesis by
ICK also depends on the level of kinase activity. This is supported by work showing that a form of ICK that is
resistant to activation, ICK
T157A
, is a weak suppressor of ciliogenesis when compared with ICK
WT
(23). It is
therefore likely that the loss of kinase activity underlies the ciliary phenotype in R05-024A.
Proteins destined for the cilia must enter through the ciliary gate, a structure at the transition zone
between the basal body and the ciliary axoneme (40) where a sorting mechanism prevents the entry of
membrane vesicles and ribosomes and guards against uncontrolled diffusion of cytosolic proteins into cilia (41).
Fig. 4E shows that the majority of ICK
WT
expressed in NIH3T3 cells localized to the basal body, whereas the
majority of mutant ICK
E80K
or ICK
R272Q
localized to the cilia tip. A similar pattern of ICK localization was found
in cells expressing ICK
WT
or the kinase-dead mutants ICK
K33R
and ICK
TDY
(29). Collectively, this evidence
suggests that ICK normally localizes to the basal body and this localization depends on kinase activity. In
addition, the observation that ICK
R272Q
failed to enter both nucleus and primary cilia in most cells suggests that
the NLS motif disrupted by R272Q substitution also tags ICK for targeting to cilia. This resembles the situation
in KIF17, where substitution of the NLS motif by alanines disrupts targeting of KIF17 both to the nucleus and
cilia (42).
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
12
In mammals, primary cilia are essential for Hh pathway signal transduction (43). In the absence of Hh
stimulation, GLI3 predominates in the processed, repressor form (GLI3R). When the Hh pathway is stimulated,
processing of GLI3 stops, the GLI3R form diminishes and there is increased active full length GLI3 (GLI3FL).
Achieving the proper balance of GLI3FL to GLI3R is essential for digit specification and Hedgehog induced
signal activity and requires IFT-mediated trafficking through the primary cilium (44). Imbalance of GLI3FL to
GLI3R resulting from deficient IFT underlies the polydactyly seen in many ciliopathies (18). Our finding of
impaired GLI3 processing, the resulting increased GLI3FL to GLI3R ratio and impaired GLI1 and PTCH1
expression in R05-024A fibroblasts is consistent with previous studies of other cilia and IFT mutants: Ift88
(Tg737
∆2-3β-gal
) (45), (Ift88
null
and Ift88
hypo
) (46); Sufu (Sufu
-/-
) (47); C2cd3 (Hty) (48); Dync2h1 (Dnchc2
lln
)
(49); Ptch1 (45); Ift172 (49); Kif3a (49); Fuz (50); Ift122 (Ift122
-/-
) (51); IFT52
hypo
(46); Kif7
-/-
(52-54). Similar
to other genes responsible for cilia function, in the ICK
E80K
mutant, the imbalance in GLI3FL to GLI3R appears
to underlie the polydactyly in R05-024A.
Concomitant to impaired GLI3 processing, the area of ciliary GLI3 accumulation in R05-024A
fibroblasts was greater than that of controls, and this observation was exaggerated by SAG treatment. The
progressive, SAG-mediated accumulation of GLI3 at the ciliary tips of R05-024A fibroblasts suggests some
degree of intact anterograde transport but defective unloading at the cilia tip and/or impaired GLI3 export out of
the cilia. Although the exact mechanism of ICK-mediated regulation of cilia function and GLI3 processing
remains to be determined, some of its features are beginning to emerge. For example, modulation of ICK
activity affected the transport velocities of several IFT components, including those belonging to both
anterograde IFT-B (KIF3A, IFT20) and retrograde IFT-A (IFT43) particles, resulting in their accumulation at
ciliary tips in ICK-deficient cells (30, 34). Interestingly, loss-of-function mutations in the C. elegans ICK
homolog DYF-5 resulted in slower IFT and impaired docking and undocking of kinesin motors from IFT
particles (24). It is therefore possible that slow IFT and/or impaired undocking of cargo causes GLI3
accumulation at the tips of R05-024A cilia (55).
While we have shown that homozygosity for a mutation ICK produces a SRPS, little is known regarding
the expression of this gene in the developing skeleton. Immunohistochemistry of a newborn mouse femur
demonstrated robust expression of ICK throughout the growth plate as well as in the periosteum/perichondrium
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
13
and the primary spongiosum. Strong expression was seen in reserve and proliferating chondrocytes and its zone
of expression was more extensive than that of Indian hedgehog (IHH), for which expression is primarily in pre-
hypertrophic and early hypertrophic chondrocytes (36). Altered GLI3 processing would be predicted to have
downstream consequences in cartilage growth plate IHH signaling. This hypothesis was confirmed by the lack
of expression of IHH targets Gli1 and Ptch1 in the growth plates of the Ick
-/-
mouse.
Although we could not examine the histological appearance of R05-024A growth plate cartilage, the
growth plate defects in Ick
-/-
animals shared some resemblance to those seen in thanatophoric dysplasia (TD),
including a short proliferative zone, small proliferating and hypertrophic chondrocytes, and decreased
extracellular matrix (Fig. 7A, B) (56), suggesting that the two disorders might share some underlying
mechanism. Mutated FGFR3 causes TD via aberrant activation of ERK MAP kinase, which inhibits
chondrocyte proliferation, induces premature senescence and disturbs chondrocyte differentiation, leading to a
short and disorganized cartilage growth plate cartilage (37, 38). In intestinal epithelial cells, suppression of ICK
expression via RNA interference results in growth arrest in the G1 phase of the cell cycle, accompanied with
induction of p21
Cip1/WAF1
cell cycle inhibitor (22). Similar changes accompany FGFR3/ERK-mediated growth
arrest in chondrocytes (57), suggesting the possibility that kinase inactive ICK-E80K causes SPRS in part via
ERK-mediated inhibition of chondrocyte proliferation. Interestingly, we found increased basal ERK activation
in R05-024A patient fibroblasts compared to control cells. These data were, however, in contrast to the similar
analysis carried-out in the growth plates of Ick
-/-
animals, which showed decreased ERK phosphorylation when
compared to control growth plates (Fig. 8D). As ERK regulates terminal chondrocyte differentiation (58), the
lower ERK activation in Ick
-/-
samples might be explained by the lack of the hypertrophic chondrocytes in the
Ick
-/-
growth plates. Future experiments should aim on elucidation of the precise role of ERK in ICK signaling in
the growth plate cartilage.
Our findings that a mutation in ICK produces a form of SRPS adds to the growing number of genes that
when mutated produce this disorder. Many of the previously identified genes are components of the ciliary IFT
machinery, while ICK is a serine-threonine kinase. Another gene that is responsible for SRPS is NEK1 (59), a
member of the NIMA related kinases in mammals that regulates ciliogenesis (60). It also is a
serine/threonine/tyrosine kinase involved in cell cycle regulation and, similar to ICK, is localized in the
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
14
centrosome/basal body (61). These data contribute to an emerging theme in SRPS, suggesting that altered
signaling through phosphorylation by serine/threonine kinases represent an additional cellular mechanism by
which skeletal ciliopathies can be produced.
MATERIAL AND METHODS
Exome sequencing
Under an approved human subjects protocol, DNA was isolated and submitted to the University of Washington
Center for Mendelian Genomics for library preparation and exome sequencing. The samples were barcoded,
captured using the NimbleGen SeqCap EZ Exome Library v2.0 probe library targeting 36.5Mb of genome, and
sequenced on the Illumina GAIIx platform with 50 bp reads. Novoalign was used to align the sequencing data
to the human reference genome [NCBI build 37] and the Genome Analysis Toolkit (GATK) (62) was used for
post-processing and variant calling according to GATK Best Practices recommendations (63, 64). Average
coverage of targeted bases was 49X with 91% of targeted bases covered by at least 10 independent reads.
Variants were filtered against dbSNP137, 95 NIEHS EGP exome samples (v.0.0.8), 6503 exomes from the
NHLBI Exome Sequencing Project (ESP6500), 1000 genomes (release 3.20120430), and 40 in-house exome
samples. Mutations were further compared with known disease-causing mutations in HGMD (2012.2). Variants
were annotated using VAX (65) and mutation pathogenicity was predicted using the programs Polyphen (66),
Sift (67), Condel (68), and CADD (69). The mutation reported in this work was confirmed by bidirectional
Sanger sequencing of amplified DNA. Sequence trace files were aligned and analyzed using Geneious version
7.1.4 created by Biomatters (http://www.geneious.com/).
Cell culture, plasmid transfection and protein analyses
Cells were propagated in DMEM media supplemented with 10% FBS and antibiotics (Life Technologies,
Carlsbad, CA). For serum starvation, NIH3T3 cells and human fibroblasts were grown in the presence of 0.1%
FBS. Cells were transfected using FuGENE6 according to manufacturer’s protocol (Promega, Madison, WI).
pCMV6 vector containing C-terminally FLAG-tagged human ICK was purchased from Origene (Rockville,
MD). Site-directed mutagenesis was used to generate the ICK
E80K
and ICK
R272Q
mutants according to the
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
15
manufacturer’s protocol (Agilent Technologies, Santa Clara, CA). To obtain protein samples from mouse tibiae
and femurs, the soft tissue was carefully removed, and the proteins were extracted into the lysis buffer
(50 mM TrisHCl pH 7.4, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, and 25 mM NaF with proteinase inhibitors
and 1mM orthovanadate) on ice for one hour. The samples were cleared by centrifugation (15,000g/10min),
their concentrations were equalized using DC Protein Assay (BioRad, Hercules, CA), and the samples were then
mixed 1:1 with 2x Laemmli sample buffer. For Western blotting, cells were lysed in Laemmli sample buffer and
protein samples were resolved by SDS-PAGE, transferred onto a PVDF membrane and visualized by
chemiluminiscence (Thermo Scientific, Rockford, IL). SAG (smoothened agonist) and PD0325901 were
obtained from Tocris Bioscience (Bristol, UK). The following antibodies were used: FLAG (1:1000; Sigma-
Aldrich, St. Louis, MO; F1804); pERK
T202/Y204
(1:1000; Cell Signaling Technology, Beverly, MA; 4370, 4376),
ERK (1:1000; Cell Signaling; 9102); actin (1:1000; Cell Signaling; 3700); MEK (1:1000; Santa Cruz
Biotechnology, Santa Cruz, CA; sc-219); pMEK
S217/S221
(1:1000; Cell Signaling; 9121); C-RAF (1:1000; BD
Biosciences, San Jose, CA; 610151); pC-RAF
S338
(1:1000; Cell Signaling; 9427); GLI3 (1:1000; R&D Systems,
Minneapolis, MN; AF3690); pICK
Y159
(1:1000; Abcam, Cambridge, MA; ab138435).
Immunoprecipitation and kinase assay
HEK293T cells transfected with FLAG-tagged ICK for 11 or 24 hours were extracted in buffer containing 50
mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% NP-40, 0.1% sodium deoxycholate, 2 mM EDTA pH 8.0, 0.5 mM
DTT, supplemented with proteinase inhibitors. Extracts were immunoprecipitated with FLAG antibody and
immunocomplexes were collected on protein A/G agarose (Santa Cruz) by overnight rotation at 4°C. Cell-free
kinase assays were carried-out with immunoprecipitated ICK or 200 ng of recombinant active ERK2 (Cell
Signaling) as a kinase, and 4 µg of recombinant MBP (Sigma) as a substrate, in 25 µl of kinase buffer (50 mM
HEPES pH 7.5, 10 mM MnCl
2
, 10 mM MgCl
2
, 8 mM β-Glycerophosphate, 1 mM DTT, 0.1 mM Na
3
VO
4
, 0.1
mM PMSF) for 30 minutes at 37°C in the presence of 1 µCi
32
P-ATP (Izotop, Budapest, Hungary). Samples
were resolved by SDS-PAGE and visualized by autoradiography.
Immunocytochemistry
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
16
Cells were plated on glass coverslips in 24 well plates, transfected with 0.25-2 g of ICK plasmid for 20 hours,
fixed with 4% paraformaldehyde, blocked with 10% goat serum (Life Technologies) and incubated with the
following primary antibodies at 4°C overnight: FLAG (1:200; Sigma-Aldrich F1804), detyrosinated tubulin
(1:1000; Millipore, Billerica, MA; AB3201), acetylated α-tubulin (1:500; Life Technologies 32-2700),
polyglutamylated tubulin (1:300; Adipogen, San Diego, CA; GT335), ARL13B (1:250; Proteintech, Rosemont,
IL; 17711-1-AP), pericentrin (1:2500; Abcam ab4448), and GLI3 (1:50; R&D Systems AF3690). Secondary
antibodies were Alexa Fluor 488/594-conjugated secondary antibodies (1:500; Life Technologies). Images were
taken on an LSM700 laser scanning microscope with acquisition done using ZEN Black 2012 software. Image
data were acquired as Z-stacks of images with 0.3 m distance between neighboring z-sections. Measurements
of cilia length in 3D was conducted in ImageJ (NIH) using plugin View5D by Rainer Heintzmann (King's
College London, UK). For calculation of the GLI3 signal at the cilia tip, Z-stacks of cilia images were acquired
using 63x oil immersion objective. 3D volume analysis was not performed due to the small GLI3 signal area
allowing only 1 to 3 Z-sections to be taken. Instead, the Z-section best representing the GLI3 signal distribution
was used to measure the area size using automatic thresholding by the intermodes method in ImageJ.
Histology and immunohistochemistry
For histology, femurs from newborn mice were fixed by cryosubstitution in methanol for immunohistochemistry
(70) and PFA (4%) for histochemistry. Samples were decalcified in 10% formic acid. For
immunohistochemistry, paraffin sections were treated with citrate buffer for antigen retrieval and quenched by
peroxidase solution. The Histostain Plus kit with DAB as a chromogen (Invitrogen) was used for ICK antibody
(1:150; Sigma; HPA001113) staining. Ick
tm1a(KOMP)Mbp
mice were crossed with Rosa26-FLP1 mice which express
FLP1 recombinase under the control of Rosa26. Then the exon 6 deleted ICK null allele, Ick
-/-
, was generated by
crossing this conditional allele with EIIA-driven Cre recombinase transgenic mice. Ick
-/-
and control mouse
sections were stained with picrosirius red for 1 hour and hematoxylin for contrast.
Scanning electron microscopy
Cells were fixed in 3% glutaraldehyde (Polysciences, Warrington, USA) dissolved in 0.2 M cacodylate buffer
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
17
(Spi supplies/Structure Probe, West Chester, USA) for 2 hours at room temperature and postfixed in 1% (v/v)
osmium tetraoxide (Degussa, Hanau, Germany) for 30 minutes at 22°C. The samples were washed in 0.2 M
cacodylate buffer, dehydrated in ascending ethanol grade and dried in a Critical Point Dryer (Balzers Union
Limited, Balzers, Liechtenstein) using liquid carbon dioxide. Samples were sputtered with gold in Sputter
Coater (Balzers) and subsequently examined using scanning electron microscopy (Tescan Orsay Holding, Brno,
Czech Republic).
Structural modeling
The three-dimensional models for wild-type and ICK
E80K
were generated by template-based (PDB ID: 3PFQ)
homology modeling using the PHYRE software (71). The ICK-specific functional elements, predicted using the
NCBI Conserved Domain Database (72), were mapped onto a three-dimensional model of ICK using the
CHIMERA software (73). Template based (PDB ID: 3C4W, 1JNK) homology modeling was employed to dock
ATP into the ATP-binding site of ICK.
In situ hybridization and quantitative RT-PCR
Limbs from control or Ick
-/-
embryo (E18.5) were fixed with 4% PFA in PBS for 3 days, decalcified with 0.5M
EDTA in RNAse-free water, and embedded for cryosection. Sections were then hybridized with digoxigenin-
labeled antisense RNA probes followed by incubation in anti-Dig antibody conjugated with alkaline
phosphatase. Colorimetric reaction was carried out using NBT/BCIP as the substrate. Riboprobes for Gli1, Ihh
and Ptch1 were prepared as previously described (74, 75). Total RNA was isolated using RNeasy Mini Kit
(Qiagen, Hilden, DE) and transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche,
Basel, CH). For quantitative PCR, LightCycler® 480 SYBER Green I Master (Roche) was used together with
the following QuantiTect Primer Assays (Qiagen): Hs_PTCH1_1_SG (QT00075824), Hs_GLI1_1_SG
(QT00060501), and Hs_GAPDH_vb.1_SG (QT02504278). ∆∆CT method was used for data quantification.
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
18
ACKNOWLEDGEMENTS
We thank Dobromila Klemova and Ladislav Ilkovics for help with electron microscopy, Miriam Minarikova for
technical assistance and Jakub Pivnicka for help with data analysis.
FUNDING
This work was supported in part by NIH grants RO1 AR066124, R01AR062651, and RO1 DE019567 to D.H.C.
and D.K. Sequencing was provided by the University of Washington Center for Mendelian Genomics (UW-
CMG) and was funded by the National Human Genome Research Institute and the National Heart, Lung and
Blood Institute grant 2UM1HG006493 to Drs. Debbie Nickerson, Michael Bamshad, and Suzanne Leal.
The NIH Training Grant in Genomic Analysis and Interpretation T32 HG002536 supported S.P.T. The work
was also supported by the Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT
LH12004, LH15231), the Grant Agency of Masaryk University (0071-2013), the European Regional
Development Fund (FNUSA-ICR No.CZ.1.05/1.1.00/02.0123; CEITEC No. CZ.1.05/1.100/02.0068) and
European Union (ICRC-ERA-HumanBridge No.316345). A Career Development Grant from the European
Organization for Molecular Biology (IG2535) and the Netherlands Organization for Scientific Research (VIDI)
supported L.T. Junior researcher funds from the Faculty of Medicine MU supported M.K.B. This research was
also supported in part by NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI
Grant Number UL1TR000124 and by Korea Mouse Phenotyping Project (NRF-2014M3A9D5A01073969) of
the Ministry of Science, ICT and Future Planning through the National Research Foundation.
FIGURE LEGENDS
Figure 1. Homozygosity for a p.E80K substitution in ICK causes SRPS. (A). Chromatogram showing
homozygosity for c.238G>A, predicting the amino acid substitution p.E80K in patient R05-024A. (B-D).
Radiographs of R05-024A showing a long narrow chest with short ribs, elongated clavicles, shortened humeri
and femora, bowed and bent radii, ulnae, tibiae, fibulae, and four limb post-axial polydactyly.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
19
Figure 2. p.E80K mutation abrogates ICK kinase activity. (A). Alignment of ICK homologs demonstrates
conservation of residue E80. Conserved residues among ICK homologs are shaded in grey; E80 is highlighted in
red. (B). 3D model of the catalytic domain of ICK. ATP-binding site (red), substrate-binding site (blue), and
activation loop (grey). Substitution of the negatively charged glutamic acid (red) for the positively charged
lysine (blue) at residue 80 is predicted to alter the electrostatic potential at the ICK-ATP interface (lower right
inset), suggesting abolition of ATP-binding. (C). HEK293T cells were transfected with empty vector (control),
wild type ICK (ICK
WT
), or mutant ICK (ICK
E80K
) as indicated and the FLAG-tagged ICK proteins were purified
by FLAG immunoprecipitation (IP). Kinase activity was measured in vitro using myelin basic protein (MBP) as
substrate in the presence of [
32
P]-ATP. Phosphorylation of MBP by recombinant ERK2 (ERK) served as a
positive control for the kinase assay. The ICK
E80K
reaction shows complete loss of kinase activity as measured
by
32
P incorporation. (D). HEK293T cells transfected with either ICK
WT
, ICK
R272Q
or kinase-dead ICK
E80K
were
analyzed for ICK activation as measured by phosphorylation of Tyr
159
(belonging to the activation T
157
-D-Y
159
motif). Mutant ICK
E80K
had only minimal activity, while ICK
R272Q
retained approximately a third of Tyr
159
phosphorylation compared to ICK
WT
(right panel; normalized to ICK expression levels). (Student’s t-test,
**p0.01; n=3).
Figure 3. The p.E80K substitution alters ICK subcellular localization in HEK293T cells. ICK overexpression
resulted in typical localization patterns shown in (A) (scale bar, 10 m) (DIC, differential interference contrast).
(B). Immunohistochemistry in HEK293T revealed differences in localization between ICK
WT
(nuclear and
cytosolic), ICK
E80K
(primarily nuclear) and ICK
R272Q
(almost exclusively cytosolic). (Student’s t-test, *p0.05,
**p0.01).
Figure 4. p.E80K substitution alters ICK distribution in primary cilia. (A-G). NIH3T3 cells were transfected
with FLAG-tagged ICK
WT
, ICK
E80K
or ICK
R272Q
and starved for 24 hours. (A). Ectopic ICK expression reduced
ciliogenesis compared to non-transfected controls, with the most pronounced reduction in ICK
WT
cells
(Student’s t-test, *p0.05, **p0.01). (B). Cells that strongly overexpressed ICK typically lack primary cilia,
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
20
compared to cells with weak ICK expression, (C) (arrow). (D). FLAG-ICK
WT
primarily localized to the ciliary
base. FLAG-ICK
E80K
and -ICK
R272Q
, in contrast, primarily localized to the ciliary tip. (E). Quantification of ICK
localization demonstrates differences between ICK
WT
, ICK
E80K
and ICK
R272Q
(t-test, *p0.05, **p0.01). (F).
Most of the cells localized FLAG-ICK
WT
and -ICK
E80K
to cilia, but only minority of cilia was positive for
FLAG-ICK
R272Q
(Student’s t-test, *p0.05, **p0.01). (G). ICK
WT
transfection shortened, while ICK
E80K
elongated cilia compared with control NIH3T3 cells (t-test, **p0.01). Dots indicate individual cilia, red bars
indicate medians. Scale bars, 10 m.
Figure 5. R05-024A fibroblasts have long cilia with abnormal morphology. (A, B). Control and R05-024A
fibroblasts stained for the cilia markers acetylated α-tubulin (AcTub, red) or ARL13B (green) (scale bars, 5m).
(C). R05-024A fibroblasts produced longer cilia compared to control fibroblasts. Cilia length was measured
using the AcTub staining and ImageJ software (t-test, **p0.01). Dots indicate individual cilia, red bars indicate
medians. (D). Scanning electron microscope demonstrated ultrastructural differences, with R05-024A cilia
appearing long, gaunt, and twisted. Scale bar, 1m.
Figure 6. R05-024A fibroblasts are deficient at processing and trafficking GLI3, and transactivation of GLI1
and PTCH1 expression. (A). Lysates from R05-024A fibroblasts showed an increased ratio of full-length
(GLI3
FL
) to repressor (GLI3
R
) GLI3 in both the presence and absence of 500nM SAG. Actin served as a loading
control. (B). Cilia from R05-024A fibroblasts exhibited pronounced accumulation of GLI3 at their tips. Cells
were treated with either 500nM (4 and 12 hours) or 100nM (24 hours) SAG, and stained for GLI3, pericentrin,
acetylated α-tubulin (Ac-Tub) or polyglutamylated tubulin (Glu-Tub). The areas of GLI3 signal at the cilia tips
were quantified and plotted using ImageJ software. Dots represent individual GLI3 areas, red bars indicate
medians. (t-test, *p0.05, **p0.01). (C). R05-024A fibroblasts have elevated levels of basal GLI1 and PTCH1
expression (t-test, **p0.01; n=4). (D). Treatment with 500 nM SAG induces GLI1 and PTCH1, that is reduced
in R05-024A fibroblasts compared with control cells (t-test, *p0.05, **p0.01; n=4).
Figure 7. Growth plates of Ick
-/-
mouse have abnormal architecture and diminished Gli1 and Ptch1 expression.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
21
Histological analysis of control and Ick
-/-
mouse growth plates. (A). Picrosirius red staining of markedly short
and hypoplastic Ick
-/-
humeral growth plate cartilage compared to normal control. (B). Comparative sections of
proliferating cartilage, hypertrophic cartilage and primary bone delineated by white squares in (A). Note
shortening of the proliferative zone, loss and disorganization of columnar hypertrophic chondrocytes, smaller
appearing proliferative and hypertrophic chondrocytes, subjectively increased cellularity throughout the growth
plate, and diminished trabeculae in the primary spongiosum in Ick
-/-
growth plates. Scale bar, 50 µm. (C). RNA
in situ hybridization analysis of the proximal tibial growth plates obtained from E18.5 control and Ick
-/-
mice.
While Ihh expression does not differ significantly between Ick
-/-
and
control, the expression of Ihh target genes
Gli1 and Ptch1 is eliminated from the Ick
-/-
growth plates or adjacent perichondrium, suggesting a defective
chondrocyte response to Ihh stimulus. Scale bars, 40 µm.
Figure 8. Modulation of ERK MAP kinase activity by ICK (A). R05-024A fibroblasts accumulated
phosphorylated (p) ERK or upstream members of its signaling module (MEK and C-RAF kinases) as compared
with control fibroblasts. (B-C). Inhibition of ERK pathway with chemical inhibitor (PD0325901) did not affect
the cilia elongation phenotype in R05-024A fibroblasts (compared to control fibroblasts, Student’s t-test,
**p0.01). Dots represent individual cilia, red bars indicate medians. (D). ERK MAP kinase activity in Ick
-/-
mice was reduced. Western blot analysis of femoral or tibial bone from Ick
-/-
mutant and control littermate (P0)
was performed to measure the ERK kinase activity. The antibodies recognized the phosphorylated ERK
(pERK
T202/Y204
) or total ERK (ERK) from femoral or tibial protein extracts and showed that the relative level of
pERK was reduced in mutant compared with wildtype control.
Supplementary Figure 1. Ick expression in the mouse cartilage growth plate. (A). ICK immunohistochemistry
in P0 femoral cartilage growth plates showing levels of expression in reserve, proliferating, prehypertrophic and
hypertrophic chondrocytes and in primary spongiosum. (B). No primary antibody control in parallel section. (C-
E) represent different differentiation zones of the growth plate seen in (A): reserve (C), proliferating (D) and
hypertrophic (E) zones. Note ICK expression in reserve and proliferating chondrocytes, bone and
perichondrium/periosteum with absent or very low expression in hypertrophic chondrocytes. Arrow points to
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
22
ICK expression in the trabecular bone and arrowhead to the perichondrium/periosteum. Bars, 50 µm.
REFERENCES
1. Provot,S. and Schipani, E. (2005) Molecular mechanisms of endochondral bone development. Biochem.
Biophys. Res. Commun., 328, 658-865.
2. Bonafe, L., Cormier-Daire, V., Hall, C., Lachman, R., Mortier, G., Mundlos, S., Nishimura,
G., Sangiorgi, L., Savarirayan, R., Sillence, D. et al. (September 23, 2015) Nosology and classification
of genetic skeletal disorders: 2015 revision. Am. J. Med. Genet. A., 10.1002/ajmg.a.37365.
3. Huber,C. and Cormier-Daire,V. (2012) Ciliary disorder of the skeleton. Am. J. Med. Genet. C Semin.
Med. Genet., 160C, 165-174.
4. Beales,P.L., Bland,E., Tobin,J.L., Bacchelli,C., Tuysuz,B., Hill,J., Rix,S., Pearson,C.G., Kai,M.,
Hartley,J.et al. (2007) IFT80, which encodes a conserved intraflagellar transport protein, is mutated in
Jeune asphyxiating thoracic dystrophy. Nat. Genet., 39, 727-729.
5. Cavalcanti,D.P., Huber,C., Sang,K.H., Baujat,G., Collins,F., Delezoide,A.L., Dagoneau,N., Le
Merrer,M., Martinovic,J., Mello,M.F. et al. (2011) Mutation in IFT80 in a fetus with the phenotype of
Verma-Naumoff provides molecular evidence for Jeune-Verma-Naumoff dysplasia spectrum. J. Med.
Genet., 48, 88-92.
6. Bredrup,C., Saunier,S., Oud,M.M., Fiskerstrand,T., Hoischen,A., Brackman,D., Leh,S.M., Midtbø,M.,
Filhol,E., Bole-Feysot,C. et al. (2011) Ciliopathies with skeletal anomalies and renal insufficiency due
to mutations in the IFT-A gene WDR19. Am. J. Hum. Genet., 89, 634-643.
7. Huber,C., Wu,S., Kim,A.S., Sigaudy,S., Sarukhanov,A., Serre,V., Baujat,G., Le Quan Sang,K.H.,
Rimoin,D.L., Cohn,D.H. et al. (2013) WDR34 mutations that cause short-rib polydactyly syndrome type
III/severe asphyxiating thoracic dysplasia reveal a role for the NF-κB pathway in cilia. Am. J. Hum.
Genet., 93, 926-931.
8. Schmidts,M., Vodopiutz,J., Christou-Savina,S., Cortés,C.R., McInerney-Leo,A.M., Emes,R.D.,
Arts,H.H., Tüysüz,B., D'Silva,J., Leo,P.J. et al. (2013) Mutations in the gene encoding IFT dynein
complex component WDR34 cause Jeune asphyxiating thoracic dystrophy. Am. J. Hum. Genet., 93,
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
23
932-944.
9. Gilissen,C., Arts,H.H., Hoischen,A., Spruijt,L., Mans,D.A., Arts,P., van Lier,B., Steehouwer,M., van
Reeuwijk,J., Kant,S.G. et al. (2010) Exome sequencing identifies WDR35 variants involved in
Sensenbrenner syndrome. Am. J. Hum. Genet., 87, 418-423.
10. Mill,P., Lockhart,P.J., Fitzpatrick,E., Mountford,H.S., Hall,E.A., Reijns,M.A., Keighren,M., Bahlo,M.,
Bromhead,C.J., Budd,P. et al. (2011) Human and mouse mutations in WDR35 cause short-rib
polydactyly syndromes due to abnormal ciliogenesis. Am. J. Hum. Genet., 88, 508-515.
11. Perrault,I., Saunier,S., Hanein,S., Filhol,E., Bizet,A.A., Collins,F., Salih,M.A., Gerber,S., Delphin,N.,
Bigot,K. et al. (2012) Mainzer-Saldino syndrome is a ciliopathy caused by IFT140 mutations. Am. J.
Hum. Genet., 90, 864-870.
12. Schmidts,M., Frank,V., Eisenberger,T., Al Turki,S., Bizet,A.A., Antony,D., Rix,S., Decker,C.,
Bachmann,N., Bald,M. et al. (2013) Combined NGS approaches identify mutations in the intraflagellar
transport gene IFT140 in skeletal ciliopathies with early progressive kidney Disease. Hum. Mutat., 34,
714-724.
13. Halbritter,J., Bizet,A.A., Schmidts,M., Porath,J.D., Braun,D.A., Gee,H.Y., McInerney-Leo,A.M.,
Krug,P., Filhol,E., Davis,E.E. et al. (2013) Defects in the IFT-B component IFT172 cause Jeune and
Mainzer-Saldino syndromes in humans. Am. J. Hum. Genet., 93, 915-925.
14. McInerney-Leo,A.M., Schmidts,M., Cortés,C.R., Leo,P.J., Gener,B., Courtney,A.D., Gardiner,B.,
Harris,J.A., Lu,Y., Marshall,M. et al. (2013) Short-rib polydactyly and Jeune syndromes are caused by
mutations in WDR60. Am. J. Hum. Genet., 93, 515-523.
15. Davis,E.E., Zhang,Q., Liu,Q., Diplas,B.H., Davey,L.M., Hartley,J., Stoetzel,C., Szymanska,K.,
Ramaswami.G., Logan,C.V. et al. (2011) TTC21B contributes both causal and modifying alleles across
the ciliopathy spectrum. Nat. Genet., 43, 189-196.
16. Dagoneau,N., Goulet,M., Geneviève,D., Sznajer,Y., Martinovic,J., Smithson,S., Huber,C., Baujat,G.,
Flori,E., Tecco,L. et al. (2009) DYNC2H1 mutations cause asphyxiating thoracic dystrophy and short
rib-polydactyly syndrome, type III. Am. J. Hum. Genet., 84, 706-711.
17. Merrill,A.E., Merriman,B., Farrington-Rock,C., Camacho,N., Sebald,E.T., Funari,V.A., Schibler,M.J.,
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
24
Firestein,M.H., Cohn,Z.A., Priore,M.A. et al. (2009) Ciliary abnormalities due to defects in the
retrograde transport protein DYNC2H1 in short-rib polydactyly syndrome. Am. J. Hum. Genet., 84, 542-
549.
18. Taylor,S.P., Dantas,T.J., Duran,I., Wu,S., Lachman,R.S., University of Washington Center for
Mendelian Genomics Consortium, Nelson,S.F., Cohn,D.H., Vallee,R.B. and Krakow,D. (2015)
Mutations in DYNC2LI1 disrupt cilia function and cause short rib polydactyly syndrome. Nat.
Commun., 6, 7092.
19. Shaheen,R., Schmidts,M., Faqeih,E., Hashem,A., Lausch,E., Holder,I., Superti-Furga,A.; UK10K
Consortium, Mitchison,H.M., Almoisheer,A., et al. (2015) A founder CEP120 mutation in Jeune
asphyxiating thoracic dystrophy expands the role of centriolar proteins in skeletal ciliopathies. Hum.
Mol. Genet., 24, 1410-1419.
20. Malicdan,M.C., Vilboux,T., Stephen,J., Maglic.D., Mian,L., Konzman,D., Guo,J., Yildirimli,D.,
Bryant,J., Fischer,R. et al. (September 18, 2015) Mutations in human homologue of chicken talpid3
gene (KIAA0586) cause a hybrid ciliopathy with overlapping features of Jeune and Joubert syndromes.
J. Med. Genet., 10.1136/ jmedgenet-2015-103316.
21. Alby,C., Piquand,K., Huber,C., Megarbané,A., Ichkou,A., Legendre,M., Pelluard,F., Encha-Ravazi,F.,
Abi-Tayeh,G., Bessières,B. et al. (2015) Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging
from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome. Am. J. Hum. Genet., 97, 311-318.
22. Fu,Z., Kim,J., Vidrich,A., Sturgill,T.W. and Cohn,S.M. (2009) Intestinal cell kinase, a MAP kinase-
related kinase, regulates proliferation and G1 cell cycle progression of intestinal epithelial cells. Am. J.
Physiol. Gastrointest. Liver Physiol., 297, G632-640.
23. Yang,Y., Roine,N. and Mäkelä,T.P. (2013) CCRK depletion inhibits glioblastoma cell proliferation in a
cilium-dependent manner. EMBO Rep., 14, 741-747.
24. Burghoorn,J., Dekkers,M.P., Rademakers,S., de Jong,T., Willemsen,R. and Jansen,G. (2007) Mutation
of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in
the cilia of Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A., 104, 7157-7162.
25. Berman,S.A., Wilson,N.F., Haas,N.A. and Lefebvre,P.A. (2003) A novel MAP kinase regulates
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
25
flagellar length in Chlamydomonas. Curr. Biol., 13, 1145-1149.
26. Bengs,F., Scholz,A., Kuhn,D. and Wiese,M. (2005) LmxMPK9, a mitogen-activated protein kinase
homologue affects flagellar length in Leishmania mexicana. Mol. Microbiol., 55, 1606-1615.
27. Lahiry,P., Wang,J., Robinson,J.F., Turowec,J.P., Litchfield,D.W., Lanktree,M.B., Gloor,G.B.,
Puffenberger,E.G., Strauss,K.A., Martens,M.B. et al. (2009) A multiplex human syndrome implicates a
key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems.
Am. J. Hum. Genet., 84, 134-147.
28. Oud,M.M., Bonnard,C., Mans,D.A., Altunoglu,U., Tohari,S., Ng,A.Y., Eskin,A., Lee,H., Rupar,C.A.,
de Wagenaar,N.P., et al. (2016) A novel ICK mutation causes ciliary disruption and lethal endocrine-
cerebro-osteodysplasia syndrome. Cilia 5, doi: 10.1186/s13630-016-0029-1
29. Moon,H., Song,J., Shin,J.O., Lee,H., Kim,H.K., Eggenschwiller,J.T., Bok,J. and Ko,H.W. (2014)
Intestinal cell kinase, a protein associated with endocrine-cerebro-osteodysplasia syndrome, is a key
regulator of cilia length and Hedgehog signaling. Proc. Natl. Acad. Sci. U. S. A., 111, 8541-8546.
30. Chaya,T., Omori,Y., Kuwahara,R. and Furukawa,T. (2014) ICK is essential for cell type-specific
ciliogenesis and the regulation of ciliary transport. EMBO J., 33, 1227-1242.
31. Fu,Z., Larson,K.A., Chitta,R.K., Parker,S.A., Turk,B.E., Lawrence,M.W., Kaldis,P., Galaktionov,K.,
Cohn,S.M., Shabanowitz,J. et al. (2006) Identification of yin-yang regulators and a phosphorylation
consensus for male germ cell-associated kinase (MAK)-related kinase. Mol. Cell. Biol., 26, 8639-8654.
32. Fu,Z., Schroeder,M.J., Shabanowitz,J., Kaldis,P., Togawa,K., Rustgi,A.K., Hunt,D.F. and Sturgill,T.W.
(2005) Activation of a nuclear Cdc2-related kinase within a mitogen-activated protein kinase-like TDY
motif by autophosphorylation and cyclin-dependent protein kinase-activating kinase. Mol. Cell. Biol.,
25, 6047-6064.
33. Wang,L.Y. and Kung,H.J. (2012) Male germ cell-associated kinase is overexpressed in prostate cancer
cells and causes mitotic defects via deregulation of APC/C-CDH1 Oncogene, 31, 2907-2918.
34. Broekhuis,J.R., Verhey,K.J. and Jansen,G. (2014) Regulation of cilium length and intraflagellar
transport by the RCK-kinases ICK and MOK in renal epithelial cells. PLoS One., 9, e108470.
35. Chen,J.K., Taipale,J., Young,K.E., Maiti,T. and Beachy,P.A. (2002) Small molecule modulation of
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
26
Smoothened activity. Proc. Natl. Acad. Sci. U. S. A., 99, 14071-14076.
36. Kronenberg,H.M. (2006) PTHrP and skeletal development. Ann. N. Y. Acad. Sci., 1068, 1-13.
37. Krejci,P., Salazar,L., Goodridge,H.S., Kashiwada,T.A., Schibler,M.J., Jelinkova,P., Thompson,L.M.
and Wilcox,W.R. (2008) STAT1 and STAT3 do not participate in FGF-mediated growth arrest in
chondrocytes. J. Cell Sci., 121, 272-281.
38. Krejci,P., Prochazkova,J., Smutny,J., Chlebova,K., Lin,P., Aklian,A., Bryja,V., Kozubik,A. and
Wilcox,W.R. (2010) FGFR3 signaling induces a reversible senescence phenotype in chondrocytes
similar to oncogene-induced premature senescence. Bone, 47, 102-110.
39. He,M., Subramanian,R., Bangs,F., Omelchenko,T., Liem,K.F.Jr., Kapoor,T.M. and Anderson,K.V.
(2014) The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium
tip compartment. Nat. Cell Biol., 16, 663-672.
40. Rosenbaum,J.L. and Witman,G.B. (2002) Intraflagellar transport. Nat. Rev. Mol. Cell Biol., 3, 813-825.
41. Pazour,G.J. and Bloodgood,R.A. (2008) Targeting proteins to ciliary membrane. Curr. Top. Dev. Biol.,
85, 115-149.
42. Dishinger,J.F., Kee,H.L., Jenkins,P.M., Fan,S., Hurd,T.W., Hammond,J.W., Truong,Y.N., Margolis,B.,
Martens,J.R. and Verhey,K.J. (2010) Ciliary entry of the kinesin-2 motor KIF17 is regulated by
importin-beta2 and RanGTP. Nat. Cell. Biol. 12, 703-710
43. Wong,S.Y. and Reiter,J.F. (2008) The primary cilium at the crossroads of mammalian hedgehog
signaling. Curr. Top. Dev. Biol., 85, 225-260.
44. te Welscher,P., Zuniga,A., Kuijper,S., Drenth,T., Goedemans,H.J., Meijlink,F. and Zeller,R. (2002)
Progression of vertebrate limb development through SHH-mediated counteraction of GLI3. Science,
298, 827-830.
45. Haycraft,C.J., Zhang,Q., Song,B., Jackson,W.S., Detloff,P.J., Serra.R. and Yoder,B.K. (2007)
Intraflagellar transport is essential for endochondral bone formation. Development, 134, 307-316.
46. Liu,A., Wang,B. and Niswander,L.A. (2005) Mouse intraflagellar transport proteins regulate both the
activator and repressor functions of Gli transcription factors. Development, 132, 3103-3111.
47. Jia,J., Kolterud,A., Zeng,H., Hoover,A., Teglund,S., Toftgård,R. and Liu,A. (2009) Suppressor of Fused
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
27
inhibits mammalian Hedgehog signaling in the absence of cilia. Dev. Biol., 330, 452-460.
48. Hoover,A.N., Wynkoop,A., Zeng,H., Jia,J., Niswander,L.A. and Liu,A. (2008) C2cd3 is required for
cilia formation and Hedgehog signaling in mouse. Development, 135, 4049-4058.
49. Huangfu,D. and Anderson,K.V. (2005) Cilia and Hedgehog responsiveness in the mouse. Proc. Natl.
Acad. Sci. U. S. A., 102, 11325-11330.
50. Heydeck,W., Zeng,H. and Liu,A. (2009) Planar cell polarity effector gene Fuzzy regulates cilia
formation and Hedgehog signal transduction in mouse. Dev. Dyn., 238, 3035-3042.
51. Cortellino,S., Wang,C., Wang,B., Bassi,M.R., Caretti,E., Champeval,D., Calmont,A., Jarnik,M.,
Burch,J., Zaret, K.S. et al. (2009) Defective ciliogenesis, embryonic lethality and severe impairment of
the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport
gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4. Dev. Biol., 325, 225-
237.
52. Cheung,H.O., Zhang,X., Ribeiro,A., Mo,R., Makino,S., Puviindran,V., Law,K.K., Briscoe,J. and
Hui,C.C. (2009) The kinesin protein Kif7 is a critical regulator of Gli transcription factors in
mammalian hedgehog signaling. Sci. Signal., 2, ra29.
53. Endoh-Yamagami,S., Evangelista,M., Wilson,D., Wen,X., Theunissen,J.W., Phamluong,K., Davis,M.,
Scales,S.J., Solloway,M.J., de Sauvage,F.J. et al. (2009) The mammalian Cos2 homolog Kif7 plays an
essential role in modulating Hh signal transduction during development. Curr. Biol., 19, 1320-1326.
54. Liem,K.F.Jr., He,M., Ocbina,P.J. and Anderson,K.V. (2009) Mouse Kif7/Costal2 is a cilia-associated
protein that regulates Sonic hedgehog signaling. Proc. Natl. Acad. Sci. U. S. A., 106, 13377-13382.
55. Tukachinsky,H., Lopez,L.V. and Salic,A. (2010) A mechanism for vertebrate Hedgehog signaling:
recruitment to cilia and dissociation of SuFu-Gli protein complexes. J. Cell Biol., 191, 415-428.
56. Wilcox,W.R., Tavormina,P.L., Krakow,D., Kitoh,H., Lachman,R.S., Wasmuth,J.J., Thompson,L,M, and
Rimoin,D,L, (1998) Molecular, radiologic, and histopathologic correlations in thanatophoric dysplasia.
Am. J. Med. Genet., 78, 274–281.
57. Krejci,P. (2014) The paradox of FGFR3 signaling in skeletal dysplasia: why chondrocytes growth arrest
while other cells over proliferate. Mutat. Res. Rev. Mutat. Res., 759, 40-48.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
28
58. Chen,Z., Yue,S.X., Zhou,G., Greenfield,E.M., and Murakami,S. (2015) ERK1 and ERK2 regulate
chondrocyte terminal differentiation during endochondral bone formation. J. Bone Miner. Res. 30, 765-
774.
59. Thiel,C., Kessler,K., Giessl,A., Dimmler,A., Shalev,S.A., von der Haar,S., Zenker,M., Zahnleiter,D.,
Stöss,H., Beinder,E. et al. (2011) NEK1 mutations cause short-rib polydactyly syndrome type majewski.
Am. J. Hum. Genet., 88, 106-114.
60. White,M.C. and Quarmby,L.M. (2008) The NIMA-family kinase, Nek1 affects the stability of
centrosomes and ciliogenesis. BMC Cell Biol., 9, 29.
61. Letwin,K., Mizzen,L., Motro,B., Ben-David,Y., Bernstein,A. and Pawson,T. (1992) A mammalian dual
specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in
meiotic germ cells. EMBO J., 11, 3521-3531.
62. McKenna,A., Hanna,M., Banks,E., Sivachenko,A., Cibulskis,K., Kernytsky,A., Garimella,K.,
Altshuler,D., Gabriel,S., Daly,M. et al. (2010) The Genome Analysis Toolkit: a MapReduce framework
for analyzing next-generation DNA sequencing data. Genome Res., 20, 1297-1303.
63. DePristo,M.A., Banks,E., Poplin,R., Garimella,K.V., Maguire,J.R., Hartl,C., Philippakis,A.A., del
Angel,G., Rivas,M.A., Hanna,M. et al. (2011) A framework for variation discovery and genotyping
using next-generation DNA sequencing data. Nat. Genet., 43, 491-498.
64. Van der Auwera,G.A., Carneiro,M.O., Hartl,C., Poplin,R., Del Angel,G., Levy-Moonshine,A.,
Jordan,T., Shakir,K., Roazen,D., Thibault,J. et al. (2013) From FastQ data to high confidence variant
calls: the Genome Analysis Toolkit best practices pipeline. Curr. Protoc. Bioinformatics, 11, 11.10.1-
11.10.33.
65. Yourshaw,M., Taylor,S.P., Rao,A.R., Martín,M.G. and Nelson,S.F. (2015) Rich annotation of DNA
sequencing variants by leveraging the Ensembl Variant Effect Predictor with plugins. Brief Bioinform.,
16, 255-264.
66. Adzhubei,I.A., Schmidt,S., Peshkin,L., Ramensky,V.E., Gerasimova,A., Bork,P., Kondrashov,A.S. and
Sunyaev,S.R. (2010) A method and server for predicting damaging missense mutations. Nat. Methods,
7, 248-249.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
29
67. Kumar,P., Henikoff,S. and Ng,P.C. (2009) Predicting the effects of coding non-synonymous variants on
protein function using the SIFT algorithm. Nat. Protoc., 4, 1073-1081.
68. González-Pérez,A. and López-Bigas,N. (2011) Improving the assessment of the outcome of
nonsynonymous SNVs with a consensus deleteriousness score, Condel. Am. J. Hum. Genet., 88, 440-
449.
69. Kircher,M., Witten,D.M., Jain,P., O'Roak,B.J., Cooper,G.M. and Shendure,J. (2014) A general
framework for estimating the relative pathogenicity of human genetic variants. Nat. Genet., 46, 310-
315.
70. Duran,I., Marí-Beffa,M., Santamaría,J.A., Becerra,J. and Santos-Ruiz,L. (2011) Freeze substitution
followed by low melting point wax embedding preserves histomorphology and allows protein and
mRNA localization techniques. Microsc. Res. Tech., 74, 440–448.
71. Kelley,L.A. and Sternberg,M.J. (2009) Protein structure prediction on the Web: a case study using the
Phyre server. Nat. Protoc., 4, 363-371.
72. Marchler-Bauer,A., Zheng,C., Chitsaz,F., Derbyshire,M.K., Geer,L.Y., Geer,R.C., Gonzales,N.R.,
Gwadz,M., Hurwitz,D.I., Lanczycki,C.J. et al. (2013) CDD: conserved domains and protein three-
dimensional structure. Nucleic Acids Res., 41, D348-352.
73. Pettersen,E.F., Goddard,T.D., Huang,C.C., Couch,G.S., Greenblatt,D.M., Meng,E.C. and Ferrin,T.E.
(2004) UCSF Chimera - a visualization system for exploratory research and analysis. J. Comput. Chem.,
25, 1605-1612.
74. Hui,C.C. and Joyner,A.L. (1993) A mouse model of greig cephalopolysyndactyly syndrome: the extra-
toesJ mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet., 3, 241-246.
75. Milenkovic,L., Goodrich,L.V., Higgins,K.M. and Scott,M.P. (1999) Mouse patched1 controls body size
determination and limb patterning. Development, 126, 4431-4440.
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
30
Table
1
Clinical
Findings
R05-024
Gestational Age at Delivery (weeks) 38
Prenatal Findings Hydrocephaly
Birth weight (grams) 4050
Birth Length (cm) 44 cm
Apgar Scores 2,6,7
Calvarium Large anterior fontanel, 7 X
7 cm, widely split sutures,
frontal bossing
Eyes Small palpebral fissures,
fused eyelids
Nose Short, upturned with tented
alae
Mouth Microstomia
Ears Low set, posteriorly rotated,
poorly formed with absent
anti-helix formation
Neck Short
Thorax Long, narrow
horizontal ribs with some
bending
Relative long appearing
clavicles
Genitalia External genital absent
Gastrointestinal Anus present
Renal No abnormalities detected
Upper extremities Foreshortened humeri
bowed and bent radii and
ulnae, smooth metaphyseal
ends of the long bones,
postaxial polydactyly
Pelvis Long narrow ileum, smooth
acetabular roof, high ischia
Lower extremities Shortened femora, bowed
and bent tibia and femora,
smooth metaphyseal ends
of the long bones, postaxial
polydactyly
at University of California, Los Angeles on August 3, 2016http://hmg.oxfordjournals.org/Downloaded from
