Korvala et al. BMC Medical Genetics 2012, 13:26
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RESEARCH ARTICLE
Open Access
Mutations in LRP5 cause primary osteoporosis
without features of OI by reducing Wnt signaling
activity
Johanna Korvala1, Harald Jüppner2, Outi Mäkitie3, Etienne Sochett4, Dirk Schnabel5, Stefano Mora6,
Cynthia F Bartels7, Matthew L Warman8, Donald Deraska9, William G Cole10, Heini Hartikka1,11, Leena Ala-Kokko1,12
and Minna Männikkö1,13*
Abstract
Background: Primary osteoporosis is a rare childhood-onset skeletal condition whose pathogenesis has been largely
unknown. We have previously shown that primary osteoporosis can be caused by heterozygous missense mutations in
the Low-density lipoprotein receptor-related protein 5 (LRP5) gene, and the role of LRP5 is further investigated here.
Methods: LRP5 was analyzed in 18 otherwise healthy children and adolescents who had evidence of osteoporosis
(manifested as reduced bone mineral density i.e. BMD, recurrent peripheral fractures and/or vertebral compression
fractures) but who lacked the clinical features of osteogenesis imperfecta (OI) or other known syndromes linked to low
BMD. Also 51 controls were analyzed. Methods used in the genetic analyses included direct sequencing and multiplex
ligation-dependent probe amplification (MLPA). In vitro studies were performed using luciferase assay and quantitative
real-time polymerase chain reaction (qPCR) to examine the effect of two novel and three previously identified mutations
on the activity of canonical Wnt signaling and on expression of tryptophan hydroxylase 1 (Tph1) and 5hydroxytryptamine (5-Htr1b).
Results: Two novel LRP5 mutations (c.3446 T > A; p.L1149Q and c.3553 G > A; p.G1185R) were identified in two
patients and their affected family members. In vitro analyses showed that one of these novel mutations together
with two previously reported mutations (p.C913fs, p.R1036Q) significantly reduced the activity of the canonical Wnt
signaling pathway. Such reductions may lead to decreased bone formation, and could explain the bone
phenotype. Gut-derived Lrp5 has been shown to regulate serotonin synthesis by controlling the production of
serotonin rate-limiting enzyme, Tph1. LRP5 mutations did not affect Tph1 expression, and only one mutant (p.
L1149Q) reduced expression of serotonin receptor 5-Htr1b (p < 0.002).
Conclusions: Our results provide additional information on the role of LRP5 mutations and their effects on the
development of juvenile-onset primary osteoporosis, and hence the pathogenesis of the disorder. The mutations causing
primary osteoporosis reduce the signaling activity of the canonical Wnt signaling pathway and may therefore result in
decreased bone formation. The specific mechanism affecting signaling activity remains to be resolved in future studies.
Background
Idiopathic juvenile osteoporosis (IJO) without features of
osteogenesis imperfecta (OI) is a rare bone condition that
affects children and adolescents. It is thought to develop
as the initiation and efficiency of bone remodeling
* Correspondence: minna.mannikko@oulu.fi
1
Oulu Center for Cell-Matrix Research, Biocenter and Department of Medical
Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland
Full list of author information is available at the end of the article
becomes impaired, thus leading to a reduced quantity of
cancellous bone [1]. The first symptoms of IJO appear
well before puberty and the principal symptoms include
reduced bone mineral density (BMD), vertebral compression fractures and metaphyseal fractures in the long
bones. The fractures lead to bone pain and impaired
mobility [1-3]. IJO is suggested to be inherited in an
autosomal dominant manner [4]. Thus far only one gene,
namely the gene encoding the low-density lipoprotein
© 2012 Korvala et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Korvala et al. BMC Medical Genetics 2012, 13:26
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receptor-related protein 5 (LRP5), has been shown to
cause juvenile-onset osteoporosis similar to IJO [4].
LRP5 has an essential role in the Wnt signaling pathway,
since it acts as a co-receptor that binds Wnt proteins with
Frizzled-receptors [5,6]. Mutations within the gene are
known to lead to various bone disorders: gain-of-function
mutations in the LRP5 gene can cause high-bone-mass
(HBM) phenotypes in humans [7,8], whereas homozygous
loss-of-function mutations cause osteoporosis-pseudoglioma syndrome (OPPG) characterized by early-onset
osteoporosis and complications in eye development [9-11].
Similarly, transgenic mice with interrupted Lrp5 express a
low bone mass phenotype, independent of Cbfa-1, including decreased osteoblast proliferation, osteopenia and persistent embryonic eye vascularization [12]. Furthermore,
associations have also been reported between the LRP5
gene polymorphisms and bone mass and size [13-15].
LRP5 is widely expressed in most human tissues, with
greater amounts in the liver and pancreas [16]. In bone,
it is mainly expressed by the bone-forming cells, i.e.
osteoblasts, in the endosteal and trabecular bone surfaces
[7,9]. It is not known to be expressed by osteoclasts [9].
Recently, Lrp5 expressed in the murine duodenum was
shown to affect the synthesis of gut-derived serotonin (5hydroxytryptamine, i.e. 5-HT) by inhibiting expression of
the serotonin rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) [17]. Serotonin then affects bone formation,
its effect being mediated by specific 5-HT transporters in
the circulation and by binding to the 5-HT receptor 1 B
(5-Htr1b) on osteoblasts [17,18]. However, other investigators have not observed a role for gut-expressed Lrp5 in
regulating serotonin production or bone mass 19.
In the present study the role of LRP5 was explored
further in 18 pediatric patients with primary osteoporosis
without features of osteogenesis imperfecta (OI). In vitro
cell culture studies were used to examine the effects of
newly found mutations on LRP5 production, the activity
of the Wnt signaling pathway, and the expression of
Tph1 and 5-Htr1b.
Methods
Subjects
The study included eight Italian and ten German patients.
All 18 pediatric patients had been referred for recurrent
fractures of long bones, bone pain, findings of osteopenia
on imaging and/or low BMD. The diagnosis of primary
osteoporosis was based on the following criteria: I) clinical
exclusion of OI, II) exclusion of secondary causes of osteoporosis, and III) low BMD, defined as Z score below -2.0,
history of recurrent peripheral fractures (≥3 fractures)
caused by low impact trauma, and/or findings of vertebral
compression fractures on x-ray films [19,20].
The control group, comprising 51 healthy individuals,
was taken from the same geographical area as the two
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German patients who had novel LRP5 mutations. In
addition, three affected and three healthy family members of the two probands with newly found mutations
were analyzed. The study was approved by the local
ethics committees, and signed informed consent was
obtained from each subject.
Molecular analysis
DNA was extracted from EDTA blood samples using
standard procedures. The 23 exons and intronic boundaries of LRP5 were amplified using the polymerase chain
reaction (PCR) method with AmpliTaq Gold DNA polymerase (Applied Biosystems), and dimethyl sulfoxide
(DMSO) was added to the reaction mixture for exon 4.
Exons 5 and 21 were amplified using AmpliTaq Gold360
(Applied Biosystems) and exon 1 was amplified with the
GC Rich PCR kit (Roche Applied Sciences). PCR primer
sequences are available on request. Mutation analysis was
performed with direct sequencing using the ABI PRISM®
3100 Genetic Analyzer and BigDye terminator cycle
sequencing chemistry (Applied Biosystems). GenBank
accession number NG_015835.1 was used as a genomic
LRP5 reference. Mutation nomenclature is in accordance
with the guidelines by den Dunnen et al. [21], and the
cDNA and protein reference sequences used were
NM_002335.2 and NP_002326.2 (GenBank).
Samples were also screened for insertions or deletions
using Multiplex Ligation-dependent Probe Amplification
(MLPA) [22]. The MLPA analysis was performed according to MRC-Holland (Amsterdam, The Netherlands) procedure using LRP5 and control probes specifically
designed in Dr. Warman’s laboratory (Additional file 1:
Table S1). The probes target sites in LPR5 differ from the
sequence of a pseudogene containing LRP5 exons 3-9
(GenBank accession number AL022324). As a control for
detecting deletion or duplication of the whole LRP5 gene
(chromosome 11), a probe pair targeting for acetylcholinesterase (ACHE) exon 2 (chromosome 7) was used.
Probes were synthesized by Integrated DNA Technologies (Coralville, IA). Unique amplicon sizes are presented
in Additional file 2: Table S2. Amplification products
were separated on ABI-PRISM® 3100 Genetic Analyzer
(Applied Biosystems) in the Case Western Reserve Genomics Core Facility (Case Western Reserve University
School of Medicine, Cleveland, Ohio), and results were
analyzed with GeneScan® Analysis 3.7 (Applied Biosystems). Peak heights of amplicons were normalized for the
ACHE peak or for the LRP5 exon 15.
In vitro studies
Constructs for in vitro studies
The wild type construct of full-length LRP5 with a carboxyl mycHis-tag (WT-LRP5-mycHis) was received from
Dr. Warman’s laboratory, having been created as
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described by Ai et al. [23]. The mutations identified in
the osteoporosis patients were introduced into the WTLRP5-mycHis construct using the QuikChange XL sitedirected mutagenesis kit (Stratagene) and the resulting
constructs were sequenced to confirm their correctness.
Five LRP5 mutants were created that included two
previously reported primary osteoporosis mutations
(C913fs, R1036Q) [4], the two novel mutations (L1149Q,
G1185R), and one HBM mutation G171V [13].
The reporter constructs SuperTOPflash (STF) and
b-galactosidase (b-Gal-CMV) were received from Prof.
Vainio’s laboratory and were used to detect the activity of
the canonical Wnt signaling pathway.
Expression of LRP5
Chinese Hamster Ovarian (CHO) cells were cultured
with 10% fetal bovine serum (FBS) (HyClone) in Dulbecco’s modified Eagle’s medium (DMEM) (BIOCHROM
AG), plated on 10 cm plates and transfected with 3 μg of
WT-LRP5-mycHis or mutant construct using Lipofectamine transfection reagent (Invitrogen) according to the
manufacturer’s protocol. After 48 h of transfection, the
cell medium was collected and the cells were lysed using
1% Triton-X-homogenisation buffer. A 7.5% SDS-PAGE
gel was prepared and 25 μl of medium or cell lysate and
10 μl of SDS-PAGE loading buffer were loaded on the gel
and analyzed under reducing conditions. Western blot
analysis was performed using the Anti-Myc tag, clone
9E10 antibody (Upstate).
Producing Wnt3a -conditioned media
Wnt3a-conditioned medium was produced and collected
from mouse L1 cells expressing Wnt3a (L Wnt3a; ATCC
CRL-2647) according to the manufacturer’s instructions.
The control-conditioned medium (L1-CM) was prepared
from a normal L1 cell line (ATCC CRL-2648) using the
same protocol as for the L Wnt3a cells.
Luciferase gene reporter assay
LRP5 mutants and 10% FBS-DMEM medium CHO
cells were plated at 2 × 104 cells/well onto a 24-well plate
and transfected 24 h later using Lipofectamine (Invitrogen). To study the effect of LRP5 mutants on Wnt signaling activity, the WT-LRP5 or mutant LRP5 construct
(20 ng), STF (100 ng) and b-Gal-CMV (5 ng) were cotransfected into cells. The total amount of transfected
DNA was adjusted to 250 ng/well by adding pcDNA3.1+
vector. Five hours later 10% FBS-DMEM was added to
each well, and the cells were collected after a further
48 h. Each transfection was performed in triplicate and
repeated at least three times on separate occasions.
LRP5 mutants and Wnt3a -conditioned medium In
experiments where Wnt3a was used to induce the activity of the pathway the transfections were performed as
described above but with 300 μl of Wnt3a-CM or control L1-CM and 200 μl of 10% FBS-DMEM added to
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each well 5 h after transfection. The cells were collected
48 h after transfections.
Measurement of luciferase activity Cell culture lysis
reagent (CCLR) was used to lyse the cells according to
the manufacturer’s instructions (Promega). The luciferase
(STF) activity was measured in a solution of 10 μl of cell
lysate and 50 μl of Luciferase Assay Reagent (Promega)
and the activity of b-galactosidase using 10 μl of cell
lysate and 70 μl of 1 × CPRG substrate (chlorophenol
red-b-D-galactopyranoside) according to the Stratagene
instructions. In both cases a VictorTM3 V 1420 Multilabel
Counter (Perkin Elmer) was used, and the relative luciferase unit (RLU) was determined from the ratio between
the luciferase and b-galactosidase activities.
Quantitative real-time polymerase chain reaction (qPCR)
The effect of LRP5 mutants on the expression of Tph1 and
5-Htr1b was studied by qPCR. CHO cells were plated at 1
× 105 cells/well on 6-well plates and transfected 24 h later
using Lipofectamine (Invitrogen) with 2 μg of either WTLRP5, the mutant LRP5 construct or pcDNA3.1+. Four
hours later 10% FBS-DMEM or Wnt3a-CM or control L1CM was added to each well (see detailed description of
CM production above), and cells were collected 48 h after
transfection. Each experiment was performed in triplicate
and repeated at least three times on separate occasions.
Total RNA was isolated using the E.Z.N.A. RNA isolation kit including a treatment with RNase-free DNase
(OMEGA Bio-Tek). RNA concentrations were measured
using a NanoDrop™ ND-1000 spectrophotometer
(Thermo Scientific) and cDNA was synthesized by reverse
transcript PCR (RT-PCR) from 1 μg of extracted RNA
using the iScript™ cDNA Synthesis kit (BioRad). Quantitative real-time PCR (qPCR) analyses of Tph1 and 5-Htr1b
expression were performed with specifically designed murine primers (available on request), and the Tph1 and
5-Htr1b results were compared with those for a standard
b-actin control. The qPCR reactions were performed
using iTaq™ SYBR Green Supermix with ROX (BioRad)
in a Mx3005P QPCR instrument (Stratagene), and the
data were assessed using MxPRo - Mx3005P v4.10 software (Stratagene). The gene expression change upon treatment was presented as relative expression (fold relative to
the non-treated control, i.e. pcDNA3.1+ transfected sample) after normalizing to b-actin, and was calculated by the
2-∆∆CT method [24]. Student’s t test was used to compare
the expression of Tph1 and 5-Htr1b in samples transfected
with LRP5 mutants with expression in samples transfected
with WT-LRP5.
Statistical analysis
Statistical analyses were performed using Student’s t
test. Values of p < 0.05 were considered statistically
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significant. Results are presented as mean ± standard
deviation (SD).
Results
Altogether 18 patients were analyzed for LRP5 gene mutations by sequencing and MLPA. No changes were
observed by MLPA (data not shown). Two new heterozygous missense mutations were found by sequencing, in
patients M11 (Figure 1A III:1) and M13 (Figure 1B II:1).
Both of these mutations, c.3446 T > A and c.3553 G > A,
were located in exon 16 of the LRP5 gene, in the fourth
YWTD/EGF domain of the LRP5 protein (Figure 2). The
mutations led to L1149Q and G1185R amino acid substitutions, respectively. Changes were also detected in
affected family members whereas neither was observed in
the non-affected family members or in the control group.
Patient M11 (Figure 1A III:1) had recurrent fractures
during childhood and adolescence and repeatedly subnormal BMD (the Z-score at lumbar spine at the time of
diagnosis was -3.20), measured by dual-energy X-ray
absorptiometry (DXA), and was diagnosed as having primary osteoporosis. M11 is currently 32 years and the
most recent BMD measurements at the lumbar spine are
normal (T-score 0.0) but osteopenic in the femoral neck
(T-score -1.5). The patient has not received bisphosphonate treatment.
The mother (Figure 1A II:2) and sister (Figure 1A
III:2) of patient M11 had the same mutation as the
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patient (c.3446 T > A; L1149Q), whereas the father (Figure 1A II:1) did not carry the mutation. In addition, the
DNA samples of maternal grandparents (Figure 1A I:1
and I:2) and maternal aunt and uncle (Figure 1A II:3
and II:4) were analyzed, but none was found to have the
mutation. Since the mother and sister have been diagnosed as having osteoporosis whereas the father shows
no signs of reduced BMD, the genetic findings are concordant with the clinical status of the patient and his
family members. The mother’s lumbar spine and
femoral neck BMD T-scores at age 48 years, after some
years of bisphosphonate treatment, were -2.3 and -1.3.
The sister’s lumbar spine and femoral neck BMD Tscores at 25 years were -1.4 and -2.5.
For the rest of the relatives, the grandmother (Figure 1A
I:1) had reduced BMD at age 83 years with lumbar spine
and hip T-scores of -2.0 and -2.2, respectively. The grandfather’s (Figure 1A I:2) BMD measurements at 83 years
were somewhat reduced (BMD T-scores of -0.9 and -2.7
for lumbar spine and hip). The aunt (Figure 1A II:3) had
osteopenia at 43 years (BMD T-scores 0.7 and -1.5 for
lumbar spine and hip). No clinical data were available for
the uncle of M11 (Figure 1A II:4). The results suggest that
M11 inherited a de novo mutation that had arisen in his
mother.
Patient M13 (Figure 1B II:1) was first presented at 13
years of age with multiple fractures in his shoulder, forearms and upper arms. The first fracture occurred at
Figure 1 A) The pedigree of patient M11, and a chromatogram showing the missense mutation c.3446 T > A (L1149Q), B) The
pedigree of patient M13, and a chromatogram showing the missense mutation c.3553 G > A (G1185R). Affected individuals carrying the
mutation are marked in black and the mutations are pointed out by means of arrows.
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Figure 2 Schematic presentation of the protein structure and domain organization of LRP5. Mutations associated with primary
osteoporosis are marked above the protein structure, and the two novel mutations found in the present study (L1149Q, G1185R) are underlined.
7 years, and he has sustained altogether 8 fractures by
age 14.5 years. When examined at the age of 13, the
patient’s alkaline phosphatase, parathyroid hormone,
25-hydroxyvitamin D 3, calcium, phosphate and creatinine levels were normal and renal function was normal.
Radiographs of the spine revealed reduced mineralization but no vertebral compression fractures or structural
abnormalities. Peripheral quantitative computed tomography (pQCT; Stratec QCT 900) of the distal radius
showed reduced total and trabecular volumetric BMD
Z-scores of -1.5 and -2.8. The lumbar spine BMD Zscore, measured by DXA, was -2.4. The diagnosis of
osteoporosis was confirmed by bone biopsy, which
showed reduced bone volume and loss of trabecular
connectivity, consistent with osteoporosis.
The father (Figure 1B I:2) of patient M13 (Figure 1B II:1)
shared the mutation (c.3553 G > A; G1185R) with his son,
but the change was not detected in the patient’s unaffected
mother (Figure 1B I:1) or sister (Figure 1B II:2). The father
has slightly reduced BMD on DXA (T-score at lumbar
spine -0.3 and at femoral neck -1.7). Otherwise the family
has a negative history of osteoporosis.
Neither one of the novel mutations (L1149Q, G1185R)
was detected in controls. The evolutionary importance of
the observed disease-causing missense mutations was
assessed by aligning the human protein sequences with
the corresponding sequences of other species. Both the
newly found mutations encode for conserved amino acids
(Additional file 3: Figure S1).
In vitro studies
Examination of the effect of the observed LRP5 gene
mutations on protein expression using SDS-PAGE and
Western blotting revealed no changes in expression levels
(data not shown).
The effect of the LRP5 mutations on the activity of the
Wnt signaling pathway was studied by comparing the
effect of the transfected mutant LRP5s with that of the
WT-LRP5. These studies were performed in the presence of 10% FBS-DMEM, Wnt3a-CM or L1-CM, and
the results were presented as fold changes relative to
the results of 10% FBS-DMEM (Figure 3). As expected,
Wnt3a-CM increased the activity of the pathway in all
the LRP5 constructs, varying from 4 (C913fs) to 9-fold
(G171V) as compared with the corresponding constructs
when using 10% FBS-DMEM (Figure 3). The addition of
Wnt3a-CM also brought out differences between the
effects of the LRP5 constructs that were not seen in the
samples treated with 10% FBS-DMEM or L1-CM. The
HBM mutation G171V showed similar activity of the
pathway to that of WT-LRP5, while a tendency for a
decrease in activity was detected with mutations C913fs,
R1036Q and L1149Q, the effects of C913fs and L1149Q
being statistically significant (p = 0.0023 and p = 0.043,
respectively) (Figure 3). The LRP5 mutant G1185R did
not affect the signaling activity.
Expression levels of Tph1 and 5-Htr1b were studied by
qPCR in CHO cells transfected with WT-LRP5 or LRP5
mutants, and in the presence of either 10% FBS-DMEM,
Wnt3a-CM or L1 control medium. Tph1 and 5-Htr1b
gene expressions were reported as fold changes relative to
the untreated control sample (pcDNA3.1+) and normalized to b-actin. The expression resulting from each LRP5
mutant in the presence of Wnt3a-CM was first compared
to the corresponding sample with L1-CM and then to
expression with WT-LRP5 (using Wnt3a-CM).
No differences were observed in the effect of the LRP5
mutants on Tph1 expression when using 10% FBS-DMEM
(data not shown), and stimulating cells with Wnt3a-CM
did not affect expression of Tph1 as compared with L1CM (Figure 4A). Only one LRP5 mutant, G1185R,
responded to Wnt3a-CM, although the difference relative
to L1-CM was still marginal (p = 0.045).
The 5-Htr1b expression was not affected by the LRP5
mutants in the presence of 10% FBS-DMEM (data not
shown), but it did increase in cells transfected with
LRP5 mutants C913fs and G1185R after the addition of
Wnt3a-CM and as compared with L1-CM (p = 0.034
and p = 0.035, respectively; Figure 4B). These 5-Htr1b
expressions did not differ from that with WT-LRP5,
however, while expression was significantly reduced in
the LRP5 mutant L1149Q (p = 0.0051 as compared with
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Figure 3 Luciferase activity in CHO cells transfected with WT-LRP5 or mutant LRP5 in the presence of Wnt3a-CM (white bars) or L1
control medium (grey bars). The activities of the signaling pathway are presented in relative luciferase units (RLU) determined by the ratio
between the luciferase and b-galactosidase activities and given as a fold change relative to corresponding samples treated with 10% FBS-DMEM.
* p < 0.05, **p < 0.01 as compared with WT-LRP5. Results are expressed as mean ± SD.
WT-LRP5; Figure 4B). Also, the R1036Q mutation
decreased 5-Htr1b expression (p = 0.02), although the
statistical significance of this effect was lost after two triplicate repeats (p = 0.50).
Discussion
We have previously identified three mutations in
the LRP5 gene that were associated with primary
osteoporosis without features of OI [4]. The present
work provides further proof of the role of LRP5 in the
disorder by revealing two additional heterozygous missense mutations (L1149Q and G1185R) in patients with
primary osteoporosis. Also, the in vitro studies showed
that the LRP5 mutations C913fs and L1149Q alter Wnt
signaling activity, as indicated by impaired activation of
LRP5 by Wnt3a.
Figure 4 A) Tph1 gene expression and B) 5-Htr1b gene expression in CHO cells transfected with wild type LRP5 (WT-LRP5) or mutant
LRP5 and treated with Wnt3a-CM or L1-CM. Expression of the Tph1 and 5-Htr1b genes is shown as a fold change relative to the untreated
control (pcDNA3.1+) and normalized to b-actin. The effects of the LRP5 mutants on the expression of these genes were examined by comparing
the results for the mutants with those for WT-LRP5 using Student’s t test. The statistical significance of each pair is denoted below the mutant
columns; **p < 0.01 as compared to WT-LRP5. Results are expressed as mean ± SD.
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All LRP5 mutations associated with primary osteoporosis in our patient set (A29T, C913fs, R1036Q, L1149Q,
G1185R) are located in the coding regions of the LRP5
gene. The L1149 and G1185 amino acids are conserved
between species (Additional file 3: Figure S1), and are
thereby likely to have structural and/or functional importance. Also, the site R1036 is quite well preserved as only
one species out of eight differs from the human sequence
at this position. The novel mutations (L1149Q and
G1185R), as well as two mutations we have identified
earlier (C913fs and R1036Q [4]), are located on the
fourth propeller domain of LRP5 protein. Only one of
the disease associated mutations (A29T) is situated in the
first propeller domain of LRP5 [4]. However, primary
osteoporosis and osteopenia have been confirmed in heterozygous carriers of OPPG-causing mutations located in
other domains and on splice sites of LRP5 [10,25]. One
of our patients (M13) with G1185R on the fourth propeller domain presented a graver phenotype than did his
father who also had the mutation. The father had
reduced BMD, but no osteoporosis. This finding is congruent with our previous and yet unpublished results
([4], Korvala et al. unpublished data) showing that phenotypes of affected offspring tend to be more severe than
those of their parents. Reasons accounting for this may
be variations in mutation penetrance or presence of
other predisposing genetic factors [26,27] or the disorder
may be multigenic in nature [28].
There is an interesting connection between the location
of LRP5 mutations and resulting disorders (and presumably the disease causing mechanisms). HBM mutations are
located in the first propeller domain of LRP5 whereas
OPPG causing mutations are scattered mainly in the second and third propeller domains. Furthermore, different
LRP5 domains bind to certain ligands in the Wnt signaling
pathway: the first and second propeller domains of LRP5/6
participate in binding a certain class of the inducing
ligands of the pathway e.g. Wnt1 and Wnt9b [29-31], but
also the Wnt signaling inhibitors Wise, Sclerostin (SOST)
and Dkk1 [31-33]. At the same time, the third and fourth
propeller domains bind DKK1 [34,35] and another class of
Wnt proteins e.g. Wnt3a (in LRP6) [30,31] while the cytoplasmic domain binds Axis inhibitor-1 (Axin) [36]. Moreover, the latest studies have indicated that different Wnts
are able to bind to specific LRP6 propellers simultaneously
[31], and compete with DKK1 binding [31,37]. These
results may potentially be implicated also in LRP5. In conclusion, the site of mutation may be an important indicator for the resulting disorder, when assuming that the
mutation affects the interaction between LRP5 and the
ligand binding the mutation site.
Our in vitro studies with four LRP5 mutations causing
primary osteoporosis showed that all the LRP5 constructs
were able to mediate signaling and that the signaling
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activity was enhanced several-fold in all the constructs
when Wnt3a was added (Figure 3), supporting the role of
Wnt3a as a ligand for LRP5. Wnt3a also enabled us to
elucidate the differences in signaling response between
the LRP5 constructs: mutants C913fs and L1149Q
reduced activity significantly (by 47% and 29%, respectively) as compared with WT-LRP5, and activity was also
reduced by R1036Q (by 24%), whereas G1185R had no
effect on signaling activity. Although no clinical significance has been reported for R1036Q (GenBank
rs61889560), it has been detected in four OPPG patients
[38] since its identification in a patient with primary
osteoporosis [4], supporting its role in bone development
or maintenance. Further functional studies are necessary
for G1185R as no effect was detected using the current
methods and a different experimental approach may
identify the underlying mechanism.
The fact that the signaling activity of HBM mutation
G171V in our study was close to that of WT-LRP5 is
consistent with earlier findings that HBM-LRP5’s are not
constitutively active but need a Wnt ligand to be activated [39-41]. In vitro LRP5 studies by others have
focused on HBM mutations and the few studies addressing the impact of mutations causing osteoporosis have
mainly been associated with OPPG. These have shown
that mutations causing OPPG reduce Wnt and/or Norrin
signaling [11,38,42], while some mutants are trafficked
unequally to the cell membrane [23]. Crabbe et al. [43]
concluded that mutations associated with idiopathic
osteoporosis in adult men may change the expression of
LRP5 protein and/or interfere with the interaction of
LRP5 with Mesd or with the Wnt/Fzd complex. Saarinen
et al. [38] found an association between three homozygous OPPG mutations (R570W, R925C, R1036Q) and
glucose tolerance, and suggested a potential association
with diabetes. Taken together, our findings are in line
with the results of other in vitro LRP5/OPPG studies
showing that mutations associated with low bone mass
disorders reduce the ability of LRP5 to mediate Wntinduced signaling and consequently result in a low bone
mass phenotype.
Since Yadav et al. [17] have shown that Lrp5 produced
in the intestine can inhibit Tph1 expression, and consequently also 5-HT synthesis and bone formation, we
examined whether the LRP5 mutations causing primary
osteoporosis influence Tph1 and/or 5-Htr1b expression in
an in vitro system. Our results showed that only one of
the mutations (L1149Q) reduced 5-Htr1b expression significantly in the presence of Wnt3a (p < 0.002; Figure 4B),
but neither HBM nor primary osteoporosis LRP5 mutations influenced Tph1 expression (Figure 4A). We cannot
readily compare our in vitro results to the in vivo studies
of Yadav et al. [17], and although the 5-Htr1b finding is of
potential interest, it is still tentative and further
Korvala et al. BMC Medical Genetics 2012, 13:26
http://www.biomedcentral.com/1471-2350/13/26
investigation using alternative methods is needed to examine its biological significance. One restriction of the current study is the use of only one reference gene, b-actin,
which has been commonly used as a reference gene in
human and murine gynecological tissue studies [44-47]
and has shown stable expression in human endometrium
[48].
The effect of 5-HT in regulating bone formation [17]
still has some open questions as discussed by Warden et
al. [18]. This is also illustrated by opposing results of Cui
et al. [49] who showed that osteocyte specific activation
or inactivation of Lrp5 in mice causes high or low bone
mass, respectively [49]. Furthermore, the bone mass of
these mice did not correlate with circulating serum serotonin levels nor did the bone markers or bone mass of
ovariectomised mice change upon treatment with Tph1
inhibitor which still resulted in decreased circulating 5HT [49]. Hence, the role of Wnt signaling pathway in
bone cannot be totally overlooked. It is supported by
both LRP5 studies [23,38,42], and by bone pathologies
caused by mutations in other components of the pathway
(e.g. SOST and DKK1). SOST mutations lead to severe
HBM disorders, sclerosteosis and van Buchem disease
[50,51], while DKK1 is shown to associate with bone
lesions in multiple myeloma [52,53]. Taken together the
studies describe the complexity of bone biology that we
are only starting to understand and unravel.
Conclusions
We have shown here that mutations causing juvenileonset primary osteoporosis reduce the signaling activity
of the canonical Wnt signaling pathway and may therefore result in decreased bone formation. Our preliminary results show reduced signaling in primary
osteoporosis mutants but the specific mechanism affecting signaling activity remains to be resolved. Since the
pathogenesis of primary osteoporosis has been largely
unknown, our results provide additional information on
the role of LRP5 mutations and their effects on the
development of this disorder.
Additional material
Additional file 1: Table S1. Probes for MLPA of LRP5 and the gene
control Acetylcholinesterase (ACHE). LRP5 probes were carefully designed
not to overlap with a pseudogene (GenBank accession number
AL022324) covering the exons 3-9 of LRP5.
Additional file 2: Table S2. Expected amplicon sizes for LRP5 and ACHE
(exon 2) in MLPA presented in increasing size order.
Additional file 3: Figure S1. Partial alignment of the human LRP5
protein sequence with Pan troglodytes, Bos taurus Mus musculus, Rattus
norvegicus, Gallus gallus, Danio rerio, Drosophila melanogaster and
Anopheles gambiae. The sites for three missense mutations associated
with primary osteoporosis are shown within boxes. Lines show lacking
sequence, and the arrowheads below the alignments point to sequence
variations.
Page 8 of 10
Acknowledgements
We would like to thank all the patients and their family members for
participating in the study. We thank Antti Railo, Ph.D., Minna Komu, Ph.D.,
Mari Taipale, M. Sc. and Marja-Riitta Väisänen, M.D., Ph.D., for their valuable
help and advice in setting up the in vitro studies. Seppo Vainio, Prof., is
acknowledged for providing the STF and b-Gal-CMV constructs. Laboratory
technicians Aira Erkkilä and Helena Satulehto are thanked for their excellent
technical assistance. The study was supported by grants from the Finnish
Cultural Foundation (to MM, LAK and JK), the Academy of Finland (LAK), the
Alma and K.A. Snellman Foundation, Oulu, Finland (JK), the Olga and Vilho
Linnamo Foundation (JK) and the Orion-Farmos Research Foundation (JK).
Author details
1
Oulu Center for Cell-Matrix Research, Biocenter and Department of Medical
Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland.
2
Departments of Medicine and Pediatrics, Massachusetts General Hospital
and Harvard Medical School, Boston, MA, USA. 3Children’s Hospital, Helsinki
University Central Hospital and University of Helsinki, and Folkhälsan
Research Center, Helsinki, Finland. 4Hospital for Sick Children, University of
Toronto, Toronto, ON, Canada. 5Department for Pediatric Endocrinology and
Diabetes, Otto-Heubner-Centrum für Kinder- und Jugendmedizin, Charite,
University Medicine Berlin, Berlin, Germany. 6Laboratory of Pediatric
Endocrinology, BoNetwork, Division of Metabolic and Cardiovascular
Sciences, San Raffaele Scientific Institute, Milan, Italy. 7Case Western Reserve
University, Department of Genetics, Cleveland, OH, USA. 8Orthopaedic
Research Laboratories, Children’s Hospital Boston, Boston, MA, USA.
9
Department of Medicine, Winchester Hospital, Winchester, MA, USA.
10
Division of Orthopaedic Surgery, Hospital for Sick Children, University of
Toronto, Toronto, ON, Canada. 11Department of Surgery, North Karelia
Central Hospital, Joensuu, Finland. 12Connective Tissue Gene Tests,
Allentown, PA, USA. 13Department of Medical Biochemistry and Molecular
Biology, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland.
Authors’ contributions
JK and HH carried out the molecular genetic studies. JK performed the in
vitro studies and drafted the manuscript. CFB and MLW performed the MLPA
analysis and MLW provided the WT-LRP5 construct. HJ, OM, ES, DS, SM, DD,
WGC, HH, LAK and MM conceived of the study, participated in its design
and coordination and commented on the manuscript. All authors read and
approved the manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 6 June 2011 Accepted: 10 April 2012 Published: 10 April 2012
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Pre-publication history
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Cite this article as: Korvala et al.: Mutations in LRP5 cause primary
osteoporosis without features of OI by reducing Wnt signaling activity.
BMC Medical Genetics 2012 13:26.
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