Genomic deletion of a long-range bone enhancer
misregulates sclerostin in Van Buchem disease
Gabriela G. Loots,1,4,5Michaela Kneissel,2Hansjoerg Keller,2Myma Baptist,2
Jessie Chang,1,4Nicole M. Collette,4Dmitriy Ovcharenko,1Ingrid Plajzer-Frick,1and
Edward M. Rubin1,3
1Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA;2Novartis Institutes for
BioMedical Research, Bone and Cartilage, Basel, Switzerland;3DOE, Joint Genome Institute,
Walnut Creek, California 94598, USA;4Genome Biology Division, Lawrence Livermore Laboratory, Livermore, California
Mutations in distant regulatory elements can have a negative impact on human development and health, yet because
of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic
purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region
deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC
recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOSTwt) or
Van Buchem (SOSTvb?) alleles. Only the SOSTwtallele faithfully expressed high levels of human SOST in the adult bone
and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes a
SOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer
assays, we were able to identify a candidate enhancer element that drives human SOST expression in osteoblast-like
cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel
function for sclerostin during limb development. Our approach represents a framework for characterizing distant
regulatory elements associated with abnormal human phenotypes.
[Supplemental material is available online at www.genome.org. The following individuals kindly provided reagents,
samples, or unpublished information as indicated in the paper: B. Black, B. Fournier, M. Brunkow, and D. Winkler.]
Deleterious mutations in distant regulatory elements postulated
to have a dramatic impact on human development and health
have been minimally explored. This problem is in large part due
to the fact that there are no simple ways to discern regulatory
elements from nonfunctional sequences or to ascertain whether
mutant phenotypes are caused by regulatory mutations. Among
Mendelian disorders associated with noncoding mutations, only
a few cases are described that clearly link alterations in distant
cis-acting regulatory regions to the cause of the disease (Ionasescu
et al. 1996; Wang et al. 2000; Enattah et al. 2002; Lettice et al.
2003; Tsui et al. 2003), and these documented cases predomi-
nantly correspond to large chromosomal aberrations (Curtin et
al. 1985; Curtin and Kan 1988; Cimbora et al. 2000; Kleinjan et
al. 2001; Chuzhanova et al. 2003). Structural variation in the
human genome described as large-scale polymorphisms has been
recently shown to be more common than previously anticipated
(Sebat et al. 2004); therefore, the extent to which large noncod-
ing duplications and deletions have an impact on human biology
remains a largely unanswered question. In this study, we dem-
onstrate that a very important skeletal dysplasia, Van Buchem
(VB) disease, associated with a large noncoding deletion is caused
by the removal of a bone-specific distant enhancer element.
Van Buchem disease (MIM 239100) is a homozygous reces-
sive disorder (Van Hul et al. 1998; Balemans et al. 2002;
Staehling-Hampton et al. 2002) that maps to Chromosome
17p21 and results in progressive increase in bone density
(Wergedal et al. 2003). The accumulation of bone mass gives rise
to facial distortions, enlargement of the mandible and head, en-
trapment of the cranial nerves, increase in bone strength, and
excessive weight (Van Hul et al. 1998; Balemans et al. 2002;
Staehling-Hampton et al. 2002). Sclerosteosis (MIM 269500) is a
cranio-tubular hyperosteosis that is phenotypically indistin-
guishable from Van Buchem (VB) disease except that it is more
severe and occasionally displays syndactyly of the digits (Beigh-
ton et al. 1977; Balemans et al. 1999; Brunkow et al. 2001; Ham-
ersma et al. 2003), a trait absent in VB patients.
An exciting development has been the recent discovery of a
negative regulator of bone formation, sclerostin (SOST) (Bale-
mans et al. 2001; Brunkow et al. 2001), whose expression is af-
fected in both sclerosteosis and Van Buchem disease. Whereas
sclerosteosis patients carry homozygous null SOST mutations, VB
patients lack any SOST coding mutations (Staehling-Hampton et
al. 2002; Winkler et al. 2003; Van Bezooijen et al. 2004). They do,
however, carry a homozygous 52-kb noncoding deletion (vb?)
∼35 kb downstream of the SOST transcript and ∼10 kb upstream
of the downstream gene, MEOX1, on human Chromosome
17p21 (Fig. 1A; Balemans et al. 2002; Staehling-Hampton et al.
2002). The shared clinical similarities between VB and scleros-
teosis along with their strong genetic linkage to the SOST locus
on Chromosome 17q12 suggests that they are allelic, and that
the deletion in VB patients removes an enhancer element essen-
tial for directing the expression of human SOST in the adult skel-
eton. To gain insight into the mechanism by which this newly
discovered gene has an impact on bone patterning and remod-
eling in Van Buchem disease, as well as to characterize the tran-
E-mail email@example.com; fax (925) 422-2099.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/
gr.3437105. Article published online before print in June 2005. Freely available
online through the Genome Research Immediate Open Access option.
15:928–935 ©2005 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/05; www.genome.org
scriptional regulation of sclerostin, we have characterized human
BAC SOST transgenic mice carrying either a normal (SOSTwt) or
an allele with the VB-associated deletion (SOSTvb?). Only the
SOSTwtallele faithfully expresses human SOST in the adult bone
and has an impact on bone metabolism, consistent with the
model that the VB noncoding deletion removes a SOST-specific
regulatory element. By exploiting cross-species sequence com-
parisons with in vitro and in vivo enhancer assays, we have iden-
tified a potential enhancer element that drives human SOST ex-
pression in the skeletal anlage, and discovered a novel function
for sclerostin during limb development, demonstrating that this
very important skeletal dysplasia, Van Buchem disease, is caused
by the removal of bone-specific distant enhancer elements and is
allelic to sclerosteosis.
Molecular and phenotypic characterization of Van Buchem
transgenic mouse models
An ∼158-kb human BAC (RP11–209M4; SOSTwt) encompassing
the 3?-end of the DUSP3 gene, SOST, MEOX1, and the ∼90-kb
noncoding intergenic interval separating sost from the neighbor-
ing gene, MEOX1, was engineered using homologous recombi-
nation in bacteria (Lee et al. 2001) to delete the 52-kb region
missing in VB patients and to create a construct that mimics the
VB allele, SOSTvb?(Fig. 1A). Three independent founder lines of
each transgenic construct were generated using standard trans-
genic procedures (Nobrega et al. 2003). Similar to the endog-
enous mouse Sost expression and the reported human expression
(Balemans et al. 2001; Brunkow et al. 2001), all lines of SOSTwt
transgenic animals reliably expressed human SOST in the miner-
alized bone of neonatal and adult mice (skull, rib, and femur),
while all of the SOSTvb?lines had dramatically reduced levels of
human SOST mRNA expression, as determined by rtPCR and
qPCR. All lines (SOSTwtand SOSTvb?) also consistently expressed
human SOST in the adult kidney and heart. Here, we present data
on the top expressing lines from each transgenic construct,
which are referred to in the manuscript as the A and B versions of
each transgene (Fig. 1B). Expression in the lung, brain, and
spleen varied among lines, while no BAC transgenic line ex-
pressed in the liver contrary to the reported human tissue-
specific expression (Brunkow et al. 2001). These data demon-
strate that in vivo, the VB allele confers dramatically reduced
SOST expression in the adult bone and suggests that the vb?
contains essential bone-specific enhancer elements.
Sclerostin is an osteocyte-expressed negative regulator of
bone formation that is structurally most closely related to the
DAN/Cerberus family of BMP antagonists (Winkler et al. 2003;
Van Bezooijen et al. 2004). In the mouse, several members of this
family including noggin and gremlin are expressed embryoni-
cally in the developing limb (Brunet et al. 1998; Khokha et al.
2003); therefore, we examined human SOST expression in the
early mouse embryo. rtPCR analysis of RNA isolated from whole
embryos showed high levels of human SOST transgenic expres-
sion in all lines from both SOSTwtand SOSTvb?transgenic animals
(Fig. 1C). SOST expression precedes endochondral ossification,
and was detected as early as embryonic day 9.5 (E9.5). Since the
VB deletion did not have an impact on human SOST embryonic
expression, we used E10.5 embryonic RNA to quantify the level
of transgene expression in different SOSTwtand SOSTvb?trans-
genic founder lines (Fig. 1D). Comparable expression levels were
also confirmed in the kidneys of SOSTwtand SOSTvb?animals.
These data strengthen the evidence that the lack of human SOST
bone expression in SOSTvb?animals is dependent on the 52-kb
noncoding deletion, rather than reflecting an artifact due to
transgene copy number or site-of-integration position effect.
The availability of BAC transgenic animals carrying wild-
type and VB alleles also allowed us to address whether the other
gene flanking the vb? region, the transcription factor MEOX1, is
affected by removing the 52-kb noncoding region. MEOX1 has
been previously shown to be involved in skeletal myogenesis
(Mankoo et al. 2003; Petropoulos et al. 2004); therefore, it has
been uncertain whether it also plays a role in the phenotypic
outcomes of Van Buchem Disease, particularly since previous ex-
periments using human patient samples prevented researchers
from directly examining the effect of the noncoding deletion on
gene expression (Staehling-Hampton et al. 2002). We have ex-
amined MEOX1 expression in all available transgenic lines from
both SOSTwtand SOSTvb?constructs, and unfortunately were not
able to detect any significant human MEOX1 expression in adult
tissues (data not shown). Based on these results, we have con-
cluded that the 209M4 BAC does not possess sufficient MEOX1-
specific regulatory elements to recapitulate wild-type human
MEOX1 expression in the SOSTwttransgenics; therefore, our ex-
periments cannot evaluate the impact the VB deletion has on
Since lack of sclerostin causes increased bone density
(Brunkow et al. 2001), we investigated whether elevated levels of
mouse models. (A) A 158-kb human BAC (SOSTwt) spanning SOST and
MEOX1 was engineered using in vitro BAC recombination in Escherichia
coli (Lee et al. 2001) by deleting the 52-kb noncoding region missing in
VB patients (SOSTvb?). Human SOST expression was analyzed by rtPCR in
adult tissues (B), embryos (C), and measured by quantitative rtPCR in
E10.5 embryos (D) from two independent lines of each SOSTwtand
Generation and characterization of Van Buchem transgenic
Sclerostin regulation in Van Buchem disease
human sclerostin have opposite effects on bone mass. Consistent
phenotypic data have been obtained for lines A of SOSTwtand
SOSTvb?transgenic constructs (Fig. 2; Supplemental material).
Animals from these lines expressed equivalent amounts of em-
bryonic human SOST (Fig. 1D, gray shaded bars). For the rest of
the manuscript we refer to these mice as SOSTwtand SOSTvb?.
SOSTwttransgenics grew to skeletal maturity with normal body
size and weight (Fig. 2A); however, the animals displayed de-
creased bone mineral density in the appendicular and axial skel-
eton, as evaluated by dual energy X-ray absorptiometry (DEXA)
analysis (Fig. 2B). Micro-Computed-Tomography (microCT)
analysis of three-dimensional cancellous bone structures re-
vealed that the mice have decreased bone volume, trabecular
number, thickness, and increased trabecular separation (Fig. 2C).
In contrast, the bone parameters of SOSTvb?transgenics were in-
distinguishable from nontransgenic littermate controls. The ob-
served osteopenia was gene dose-dependent. SOSTwttransgenic
mice bred to homozygosity revealed a further dramatic decrease
in tibial cancellous bone volume (Fig. 3A,C). Histomorphometric
analysis revealed that these animals display further decreased
bone formation rates at skeletal maturity reflected in decreased
fluorochrome marker uptake into mineralizing bone both in can-
cellous (Fig. 3B,D) and cortical bone (tibia: non-tg = 0.319 ?
0.016 µm/d vs. SOSTwt/wt= 0.110 ? 0.027 µm/d; p < 0.001) in
both the appendicular (Fig. 3B,C) and the axial skeleton. Neither
the number of terminally differentiated bone-forming cells, the
osteocytes, nor the number of bone-resorbing cells, the osteo-
clasts, were significantly affected by the transgene expression
(data not shown).
In contrast to SOSTwttransgenics, SOSTvb?animals did not
display an osteopenic bone phenotype in either the appendicular
or the axial mature skeleton, even in the homozygous configu-
ration (Fig. 2B,C; Supplemental material). These data demon-
strate that modulation of SOST expression has a dramatic impact
on bone formation in the adult mammalian skeleton. Most im-
portantly, these phenotypic data suggest that overexpressing hu-
man SOST under the control of its own proximal promoter ele-
ments in concert with the downstream VB region negatively
modulates adult bone mass. In contrast, bone mass is unaffected
in transgenic animals that lack the 52-kb VB region, in a con-
struct that mimics the allele carried by VB patients, consistent
with the model that Van Buchem disease is caused by removing
a bone-specific regulatory element.
Interestingly, and consistent with the observed embryonic
expression, elevated levels of human SOST result in abnormal
digit development in both SOSTwtand SOSTvb?BAC transgenics
bred to homozygosity. The forelimbs and hindlimbs of these ani-
mals display a wide range of fused and missing digits as visualized
by autoradiography (data not shown) and microCT (Fig. 4B). rt-
PCR data correlate SOST expression with the severity of digit
abnormalities (data not shown). Mouse whole mount in situ hy-
bridization revealed SOST to be expressed as early as E9.5, pre-
dominantly in the mesenchymal tissue of the developing limb
bud (Fig. 4A). These findings imply that SOST embryonic expres-
sion is controlled by a transcriptional regulatory element differ-
ent from the one driving the adult bone expression, consistent
with the observation that both sclerosteosis and VB patients suf-
fer from abnormal bone mass accumulation, while only scleros-
teosis patients exhibit syndactyly of the digits (Staehling-
Hampton et al. 2002).
Comparative sequence analysis and enhancer assays
Given the striking bone phenotypes observed in both VB and
sclerosteosis patients, we next focused on the identification of
noncoding sequences required for SOST bone-specific expression
through a combination of comparative sequence analysis and
transient transfection assays. We aligned an ∼140-kb human
SOST region (RP11–209M4; AQ420215, AQ420216) (http://
zpicture.dcode.org/; Ovcharenko et al. 2004) to the correspond-
ing mouse sequences from Chromosome 11 (Mouse chr11:
101,489,231–101,688,385; Oct.03 Freeze) (Fig. 5A). A stringent
requirement of at least 80% identity (% ID) over a 200-bp win-
dow (?80% ID; ?200 bp) identified
seven evolutionarily conserved regions
(ECR2–8) within the vb? genomic inter-
val, which were prioritized for in vitro
enhancer analysis. ECR2–8 were tested
for their ability to stimulate a heterolo-
gous promoter (SV40) in osteoblastic
(UMR-106) and kidney (293) derived cell
lines. One element, ECR5, was able to
stimulate transcription in UMR106 cells
(Fig. 5B), but not in the kidney cell line,
suggesting that ECR5 enhancer function
is specific to the osteoblastic lineage. We
also tested the transcriptional activity of
the human SOST proximal promoter re-
gion (2-kb region upstream of the 5?-
UTR) in the two cell types and compared
it to the SV40 and the osteoblast-specific
osteocalcin promoter (Og2). The SV40
promoter showed comparable activity in
both cell lines and, as expected, Og2 was
only active in the UMR-106 cells. The
human SOST promoter stimulated tran-
scription in the osteoblastic cells simi-
larly, albeit with slightly higher activity
than the Og2 promoter, while it demon-
measurements of 5-mo-old male mice (non-tg = 13; SOSTwt= 15; SOSTvb?= 14; animals were pooled
from two lines of SOSTwtand two lines of SOSTvb?; Supplemental material). (B) Bone mineral density
in the tibia, femur, and lumbar spine as evaluated by DEXA. (C) Bone volume, trabecular number,
thickness, and separation as evaluated in the cancellous bone compartment of the proximal tibia
metaphysis by microCT. Mean ? SEM; (*) p < 0.05 vs. non-tg.
SOST transgenic expression has a negative impact on bone parameters. (A) Body weight
Loots et al.
930 Genome Research
strated stronger activity in kidney cells (Fig. 5B). These data sug-
gest that SOST kidney expression may be due to proximal pro-
moter sequences, whereas strong expression in osteoblast cells
requires the activity of the ECR5 element. Consistent with the
results obtained from transfecting SV40 promoter constructs,
only ECR5 was capable of activating the human SOST promoter
(4X) in UMR106 cells (Fig. 5C), while all other ECR-constructs
had background level expression. Thus, a small sequence ele-
ment within the vb? region (ECR5) was identified that confers in
vitro osteoblast-specific enhancer activity onto both the human
SOST and the SV40 heterologous promoter.
To test ECR5’s ability to drive expression in the skeletal
structures of the mouse embryo, we expressed an ECR5-hsp68-
LacZ construct in transgenic mice (Fig. 5D; Nobrega et al. 2003).
Transient transgenic animals were created using standard tech-
niques (Mortlock et al. 2003) and F0pups were stained for ?-ga-
lactosidase (LacZ) expression at E14.5 (Nobrega et al. 2003).
Transgenic embryos expressed LacZ in cartilage of the ribs, ver-
tebrae, and skull plates (Fig. 5D), and the expression was identical
in all positive transgenic embryos obtained from two indepen-
dent injections (N = 2). In parallel we also injected ECR4-hsp68-
LacZ and ECR6-hsp68-LacZ constructs and assayed LacZ expres-
sion at E12.5 and E14.5. None of the ECR4 (N = 6) and ECR6
(N = 2) positive embryos expressed LacZ at these time points.
These data highly suggest that the 250-bp ECR5 element con-
tained within the 52 kb deleted in VB patients functions to drive
SOST bone-specific expression in vivo.
Sclerosing bone dysplasias are rare genetic disorders in which
excessive bone formation occurs because of defects in bone re-
modeling (Van Hul et al. 2001). Identifying the responsible
genes, their regulation, and mechanisms of action will provide
useful insights into bone physiology and potentially benefit the
treatment of these disorders, as well as facilitate the development
of therapies for replenishing bone loss in osteoporosis. In this
study we have demonstrated that the 52-kb noncoding deletion
present in Van Buchem patients removes a distant SOST-specific
regulatory element, and therefore Van Buchem disease is hypo-
morphic to sclerosteosis. Currently, we don’t have a clear view of
how the lack of sclerostin promotes osteogenesis; therefore, elu-
cidating its transcriptional regulation is key to understanding the
interconnection between its expression pattern in osteogenic
cells and its mode of action either as a BMP-antagonist (Winkler
et al. 2003) or WNT-antagonist (Li et al. 2005). The elaborate
expression pattern we detect along with the multitude of puta-
tive enhancer elements that have the potential to have a positive
or negative impact on SOST in a spatial and temporal precise
manner attest to this molecule’s complexity and functional ver-
mal tibia metaphysis of 5-mo-old male mice (non-tg = 5; SOSTwt= 7;
SOSTwt/wt= 4). (A) Bone volume and (B) bone formation rates as deter-
mined by microCT scans and histomorphometric analysis, respectively.
Mean ? SEM; (*) p < 0.05 vs. non-tg; (X) p < 0.05 vs. SOSTwt. (C) Can-
cellous bone compartment of nontransgenic and SOSTwt/wtmice. (D)
Fluorochrome marker uptake at site of active mineralization of bone ma-
trix laid down by osteoblasts in wild-type and transgenic mice at the
interface between endocortex and cancellous bone.
Human SOST dose effect on bone metabolism in the proxi-
SOSTvb?transgenic mice. (A) High levels of embryonic SOST expression
were predominantly detected in the developing limb bud of E10.5 de-
fective mice, as visualized by whole-mount in situ hybridization using a
SOST probe that detects human and mouse transcripts. (B) microCT scans
of defective SOSTwtand SOSTvb?adult limbs overexpressing human SOST.
Embryonic sost expression and limb deformity in SOSTwtand
Sclerostin regulation in Van Buchem disease
satility. Consistent with this view, our analysis provides robust in
vivo evidence for the role of sclerostin during bone formation,
modulation of adult bone mass, and for a novel function during
In general, the osteopenic phenotype we observed is consis-
tent with reports describing transgenic mice overexpressing
BMP-antagonists from cDNA constructs driven by the osteocal-
Og2>SOST transgenic animals (Winkler et al. 2003), we never
observed architectural disorganization of the lumbar vertebrae.
We believe the osteopenic phenotypic variations between the
cDNA and BAC SOST transgenic mice are most likely attributed to
the transcriptional control of human SOST in each transgenic
construct. SOSTwtBAC transgenics more faithfully mirror the
proper regulatory control exerted on the SOST gene in the en-
dogenous context of the human genome, while the Og2>SOST
transgenic expression is ectopic and highlights the transcrip-
tional specificity of the osteocalcin promoter.
alignment generated using the zPicture alignment engine (http://zpicture.dcode.org/). Exons are in blue, untranslated regions in yellow, repetitive
elements in green, and noncoding sequences in red (intragenic) or pink (intronic). Seven highly conserved elements (?200 bp; ?80% ID; ECR2–8)
within VB? were tested in rat osteosarcoma (UMR-106) and kidney cells (293) for the ability to enhance luciferase expression from the SV40 promoter
(B) or human SOST promoter (C). ECR5 activates the human SOST promoter in rat osteosarcoma cells (C), and drives the hsp68 promoter in the skeleton
of E14.5 mouse embryos (D).
Enhancer activity of evolutionarily conserved noncoding sequences from the Van Buchem deletion region. (A) Human/mouse genomic
Loots et al.
932 Genome Research
Since sclerosteosis is caused by SOST-null mutations (Bale-
mans et al. 2001; Brunkow et al. 2001), our results indicate that
VB disease and sclerosteosis are allelic, and VB patients are hy-
pomorphic for the SOST gene and lack SOST expression in the
adult bone. Our data suggest that SOST embryonic expression is
unaltered in VB patients, who also never display syndactyly of
the digits, indicating that both reduced and elevated levels of
human sclerostin have a negative impact on limb development
and digit formation, a novel function attributed to this molecule.
Our findings provide evidence that noncoding regions in
the VB deletion control sclerostin expression levels and modulate
BMD in mice; therefore, an important question is whether varia-
tion in BMD in the general population could also be directly
impacted by sequence variants in key noncoding regions of the
VB deletion. A recent new study investigated the association be-
tween common polymorphisms in the SOST gene region with
BMD in elderly whites (Uitterlinden et al. 2004). From a set of
eight polymorphisms, one 3-bp deletion (SRP3) from the SOST
promoter region was associated with decreased BMD in women,
and a polymorphic variant (SRP9) from the VB deletion region
was associated with increased BMD in men. Whereas this SRP9
does not map on any human–mouse conserved region in the VB
deletion, an important question for future studies is whether this
SNP is in linkage disequilibrium with ECR5 or if additional func-
tional SNPs could be identified in this or other SOST-specific
The genetic factors that contribute to susceptibility to bone
loss are extremely heterogeneous; therefore, murine models that
affect bone development and growth can provide invaluable in-
sights into the molecular mechanisms of progressive bone loss in
humans. Human genetic diseases of the skeleton such as scleros-
teosis and Van Buchem disease provide a starting point for un-
derstanding the modulation of anabolic bone formation, and
ultimately have the potential to identify key molecular compo-
nents that can be used as new therapeutic agents to treat indi-
viduals suffering from bone loss disorders. Our study also pro-
vides strong support for the utilization of comparative sequence
analysis to dramatically filter through nonfunctional regions in
the human genome and enhance the discovery of noncoding
disease-causing mutations both in discrete enhancer elements or
in large noncoding deletions. This study represents a clear and
unambiguous case in which altering noncoding genomic content
has a deleterious impact on gene expression, demonstrating that
mutations in distant regulatory elements are able to cause con-
genital abnormalities analogous to coding mutations.
Generating transgenic mice
An FRT-kan-FRT cassette was excised from a pICGN21 vector
(KpnI; SacI) and inserted into pUC18 to create pUC18.kan.FRT.
Homologous arms were PCR-amplified from 209M4 BAC DNA
and cloned into a pUC18.kan.FRT vector using EcoRI/SacI sites
for the left arm (VBDelH1: fwd, 5?-TTGGTACCGGATTGAAGTGA
TCCCCAGCTGGA-3?; rvd, 5?-TTGAGCTCCAATCTCCTGACCTT
GTGATCCGC-3?), and the SmaI site for the right arm (VbDelH2:
fwd, 5?-TTCCCGGGCGCTTGAACCCAGTAGGTGGAGG-3?; rvd,
to create the recombination vector pUC18.kan.FRT.VBDel. Then
200–300 ng of KpnI-digested VBDelH1-FRT-kan-FRT-VbdelH2
fragment was electroporated into EL250–209M4 cells. Recombi-
nant BACs were identified by PCR and pulse-field gel analysis,
were isolated at a final concentration of 1 ng/mL, and microin-
jected into fertilized FVB mouse eggs using standard procedures.
Transgenic mice were genotyped using PCR analysis of DNA pre-
pared from tail DNA of founder animals using the following
primer pair: 5?-ATGTCCACCTTGCTGGACTC-3? and 5?-GTCTGT
GGGCTGGTTTGCAT-3?. Transgenic mice were maintained on
an FVB background.
RT-PCR, quantitative RT-PCR, and in situ hybridization
Total RNA was isolated with Trizol reagent (Invitrogen) and re-
verse-transcribed into cDNA (Superscript II; GIBCO) using stan-
dard methods. cDNA was amplified using a GC-Melt PCR kit
(Clontech) (65°C annealing/3 min extension/35 cycles) using hu-
man (fwd, 5?-AGAGCCTGTGCTACTGGAAGGTGG-3?, rvd, 5?-
TAGGCGTTCTCCAGCTCGGCC-3?) and mouse (fwd, 5?-GACTG
GAGCCTGTGCTACCGA-3?, rvd, 5?-CTTGAGCTCCGACTGG
TTGTGGAA-3?) SOST primer sets. Mouse ?-actin (fwd, 5?-CCTCT
ATGCCAACACAGTGC-3?, rvd, 5?-CTGGAAGGTGGACAGTGAG
G-3?) was used as control (58°C annealing/30 sec extension/25
cycles). Quantitative rtPCR expression analysis was performed
using an ABI Prism 7900HT sequence detection system, TaqMan
Universal PCR Master mix, human 18S rRNA pre-developed Taq-
Man assay reagent for normalization, and TaqMan Assay-on-
Demand products for mouse, rat, and human SOST, all from
Applied Biosystems. We considered noon on the day that we
found a vaginal plug to be E0.5. We carried out RNA localization
by whole-mount in situ hybridization according to established
protocols. RNA antisense probes were labeled with digoxigenin
and were synthesized with T7 RNA polymerase as previously de-
Dual energy X-ray absorptiometry (DEXA) analysis
Tibial, femoral, and lumbar vertebral bone mineral density (in
milligrams per square centimeter) was measured using a regular
Hologic QDR-1000 instrument (Hologic). A collimator with 0.9-
cm-diameter aperture and an ultrahigh resolution mode (line
spacing, 0.0254 cm; resolution, 0.0127 cm) was used. The excised
long bones were placed in 70% alcohol onto a resin platform
provided by the company for soft tissue calibration. Daily scan-
ning of a phantom image controlled the stability of the measure-
ments. Instrument precision and reproducibility had been previ-
ously evaluated by calculating the coefficient of variation of re-
peated DEXA and had been found to be below 2%. Coefficients of
variation were 0.5% to 2% for all evaluated parameters. A set of
5-mo-old male mice was analyzed (non-tg = 13 littermates of all
analyzed lines; SOSTwt= 15 heterozygous mice from two SOSTwt
BAC lines; SOSTvb?= 14 offspring of heterozygous matings from
Micro-Computed-Tomography (microCT) analysis
Cancellous bone structure was evaluated in the proximal tibia
metaphysis using a Scanco vivaCT20 (Scanco Medical AG). The
nonisometric voxels had a dimension of 12.5 µm ? 12.5
µm ? 12.5 µm. From the cross-sectional images the cancellous
bone compartment was delineated from cortical bone by tracing
its contour at every tenth section. In all the other slices, bound-
aries were interpolated based on the tracing to define the volume
of interest. In all, 660 slices covering a total length of 0.8 mm
within the area of the secondary spongiosa (1.3 mm from the
proximal end) were evaluated. A threshold value of 175 was used
for the three-dimensional evaluation of trabecular number,
thickness, and separation. Both sets of male 5-mo-old mice on
which DEXA and histomorphometric analysis have been per-
formed were analyzed. A voxel size of 25 µm ? 25 µm ? 25 µm
Sclerostin regulation in Van Buchem disease
was chosen for visualization of the digits of the forelimbs and
After dissection, the tibia and lumbar vertebrae were placed for
24 h in Karnovsky’s fix, dehydrated in ethanol at 4°C, and em-
bedded in methylmethacrylate. A set of 4-µm and 8-µm-thick
nonconsecutive microtome sections were cut in the frontal mid-
body plane for evaluation of fluorochrome-label-based dynamic
and cellular parameters of bone turnover. The 4-µm-thick sec-
tions were stained with TRAP and Giemsa stain. The sections
were examined using a Leica DM microscope (Leica) fitted with a
camera (SONY DXC-950P) and adapted Quantimet 600 software
(Leica). Two sections/animal were sampled for all sets of param-
eters. Microscopic images of specimens were evaluated semiau-
tomatically digitally (400? magnification). All parameters were
measured and calculated according to Paritt et al. (1987). Fluo-
rochrome label bone formation dynamics were evaluated on un-
stained 8-µm-thick sections. Bone perimeter, single- and double-
labeled bone perimeter, and interlabel width were measured.
Mineralized perimeter (%), mineral apposition rates (microme-
ters/day; corrected for section obliquity in the cancellous bone
compartment), and daily bone formation rates (daily bone for-
mation rate/bone perimeter; micrometer/day) were calculated.
Osteoclast numbers (osteoclast number/bone perimeter; per mil-
limeter) and perimeter values (osteoclast perimeter/bone perim-
eter; percent) were determined on the TRAP-stained slides, and
osteocyte number (osteocyte number/bone perimeter; per milli-
meter) on the Giemsa-stained slides. All parameters were evalu-
ated in the spongiosa and at the endocortex. A set of 5-mo-old
male mice from one SOSTwtline was analyzed (non-tg = 5,
SOSTwt= 7, SOSTwt/wt= 4).
In vitro enhancer assays
ECRs were PCR-amplified with 5?-NheI-linkers, TOPO-cloned
into a pCR2.1 vector (Invitrogen), then shuttled into NheI/XhoI
sites of a pGL3 promoter (Promega) or HindIII/PstI of hsp68-LacZ
(B. Black). The following primers were used to amplify human
DNA (62°C annealing/30 sec extension/35 cycles): ECR2 (545
bp), 5?-AGCAACGCAGGGCAGGAGCCAAGA-3?, 5?-TAGCTGGC
CTCTCCTGGGCGTCTT-3?; ECR3 (410 bp), 5?-GGGGGCTGTAT
CCT-3?; ECR4 (296 bp), 5?-TGACAAACAGGAAGGTGGCAGGGC
-3?, 5?-CCCCCAACATTCCTGTCCCCTTG-3?; ECR5 (259 bp), 5?-
TGGTCTCATTTG-3?; ECR6 (666 bp), 5?-CCCTGAGAAACATGCC
ECR7 (568 bp), 5?-AAACTGCCAAGCCCCAGCTGGCTA-3?, 5?-
GCCCAGGGCTCAGAAATGTGTGGA-3?; ECR8 (352 bp), 5?-
AATGGCTGGGG-3?; ?2 kb promoter, 5?-CAGCAGAAGATGTCA
CAGCAGG-3?, 5?-GAGCTGCATGGTACCAGCCAGA-3?. The hu-
man SOST promoter sequence (2 kb upstream of the 5?-UTR) was
PCR-amplified with SmaI linkers and transferred into the SmaI
site of pGL3basic (Promega). A luciferase reporter plasmid con-
taining the mouse osteocalcin (Og2) promoter sequence from
?1323 to +10 in pGL3basic was kindly obtained from B. Fournier
(Novartis Basel, Switzerland). Reporter plasmids containing ECR-
4, ECR-5, or ECR-6 upstream of the human SOST promoter were
generated by inserting the ECR elements into the NheI site. Plas-
mid DNA was isolated using standard endotoxin-free methods
(QIAGEN). FuGene (Roche) and a CMV-bgal reporter plasmid
(Clontech) as internal control were used for transient transfec-
tions of rat UMR-106 and human 293 cells. Cells were incubated
for 24 h at 37°C, and luciferase and galactosidase expression were
measured using standard assay kits (Promega).
Transient transgenic analysis
For transient transgenic analysis, 500 mg of DNA was linearized
with NotI, followed by CsCl gradient purification, and 2–5 ng
was used for pronuclear injections of FVB embryos. E12.5–E14.5
embryos were dissected in ice-cold PBS, and were fixed in 4%
paraformaldehyde at 4°C for 1–2 h, and stained for LacZ as de-
scribed. Transgenic embryos were detected by PCR from tail DNA
(fwd, 5?-TTTCCATGTTGCCACTCGC-3?; rvd, 5?-AACGGCTT
GCCGTTCAGCA-3?; 55°C annealing/30 sec extension/25 cycles).
The authors thank V. Afzal, M. Bruederlin, E. Kuhn, H. Jeker, M.
Merdes, A. Studer, and J. Wirsching for excellent technical help
and B. Fournier for sharing the mouse osteocalcin reporter plas-
mid. Many thanks to M. Brunkow and D. Winkler for providing
us with the original 209M4 human BAC. The authors are deeply
indebted to Lisa Stubbs, Richard Harland, Mustafa Khokha, and
Marcelo Nobrega for invaluable advice and comments on the
manuscript. G.G.L. was supported by the Department of Energy
Alexander Hollaender Fellowship and by the NIH (HD47853-01).
J.C. and N.M.C. were supported by NIH (HD47853-01). This work
was performed under the auspices of the U.S. Department of
Energy by the University of California, Lawrence Berkeley Na-
tional Laboratory, Contract No. AC0376SF00098 and Lawrence
Livermore National Laboratory Contract No. W-7405-Eng-48.
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Received November 5, 2004; accepted in revised form April 27, 2005.
Sclerostin regulation in Van Buchem disease