Normal Leptin Expression, Lower Adipogenic Ability, Decreased Leptin Receptor and Hyposensitivity to Leptin in Adolescent Idiopathic Scoliosis
Decreased Leptin Receptor and Hyposensitivity
to Leptin in Adolescent Idiopathic Scoliosis. PLoS ONE 7(5): e36648. doi:10.1371/journal.pone.0036648
Normal Leptin Expression, Lower Adipogenic Ability, Decreased Leptin Receptor and Hyposensitivity to Leptin in Adolescent Idiopathic Scoliosis
Guoyan Liang 0
Wenjie Gao 0
Anjing Liang 0
Wei Ye 0
Yan Peng 0
Liangming Zhang 0
Swarkar Sharma 0
Peiqiang Su 0
Dongsheng Huang 0
Katriina Aalto-Setala, University of Tampere, Finland
0 1 Department of Orthopedics, Sun Yat-sen Memorial Hospital of Sun Yat-sen University , Guangzhou, Guangdong , China , 2 Seay Center for Musculoskeletal Research, Texas Scottish Rite Hospital for Children, Dallas, Texas, United States of America, 3 Department of Spine Surgery, The First Affiliated Hospital of Sun Yat-sen University , Guangzhou, Guangdong , China
Leptin has been suggested to play a role in the etiology of Adolescent Idiopathic Scoliosis (AIS), however, the leptin levels in AIS girls are still a discrepancy, and no in vitro study of leptin in AIS is reported. We took a series of case-control studies, trying to understand whether Leptin gene polymorphisms are involved in the etiology of the AIS or the change in leptin level is a secondary event, to assess the level of leptin receptor, and to evaluate the differences of response to leptin between AIS cases and controls. We screened all exons of Leptin gene in 45 cases and 45 controls and selected six tag SNPs to cover all the observed variations. Association analysis in 446 AIS patients and 550 healthy controls showed no association between the polymorphisms of Leptin gene and susceptibility/severity to AIS. Moreover, adipogenesis assay of bone mesenchymal stem cells (MSCs) suggested that the adipogenic ability of MSCs from AIS girls was lower than controls. After adjusting the differentiation rate, expressions of leptin and leptin receptor were similar between two groups. Meanwhile, osteogenesis assay of MSC showed the leptin level was similar after adjusting the differentiation rate, but the leptin receptor level was decreased in induced AIS osteoblasts. Immunocytochemistry and western blot analysis showed less leptin receptors expressed in AIS group. Furthermore, factorial designed studies with adipogenesis and osteogenesis revealed that the MSCs from patients have no response to leptin treatment. Our results suggested that Leptin gene variations are not associated with AIS and low serum leptin probably is a secondary outcome which may be related to the low capability of adipogenesis in AIS. The decreased leptin receptor levels may lead to the hyposensitivity to leptin. These findings implied that abnormal peripheral leptin signaling plays an important role in the pathological mechanism of AIS.
Funding: This work was supported by the National Natural Science Foundation of China (No. 81171674); National Natural Science Foundation of China
(No. 81071703); Research Fund of Social Development of Guangdong Province (No. 2010B031900023); Research Fund of popular science of Guangzhou City
(No. 2011KP012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Adolescent idiopathic scoliosis (AIS) is a common tridimensional
deformity, characterized by rotation of the vertebrae and lateral
deviation of the spine. So far, the exact etiology of AIS remains
elusive. It is generally accepted that AIS is a systemic disease and
the scoliosis mainly results from the abnormal systemic skeletal
growth and the asynchronous spinal neuro-osseous growth [1,2,3].
Also, AIS has been observed as a complex genetic disorder, and
recent genome-wide association studies have implicated some new
candidate genes [1,2,4,5,6]. Interestingly, several studies had
found the AIS patients (especially in girls) have common features
of taller stature, lower body mass index (BMI) and systemic low
bone mass [7,8,9,10,11], which may be owing to a cytokine-like
protein hormone: leptin [3,11].
Leptin is coded by the Leptin gene (i.e. the obese gene, Ob) and is
primarily expressed in white adipose tissue. It binds to leptin
receptors and plays key roles not only in regulating the energy
intake and expenditure of the body, but also in connecting the
changes in body composition with bone formation and resorption
[12,13,14]. Leptin affects bone metabolism via central and
peripheral ways. It modulates cortical bone formation by
regulating the expression of several neuropeptides in
hypothalamus and inducing sympathetic activation [12,15,16]. It also directs
the bone marrow stromal cells to osteogenic instead of adipogenic
pathway [17,18]. Thus an abnormal leptin level or the deficiency
of signal pathway may result as a disorder in skeletal growth.
Leptin and its signaling pathway may be a candidate for the
etiology of AIS. Significantly lower serum leptin levels were found
in girls with AIS, and the leptin levels also correlated significantly
with body weight, BMI and body mineral density (BMD) .
However, recently the same group claimed that the serum total
leptin level between AIS and healthy girls are similar after
adjusting the BMI . Both of the studies were conducted with
blood samples of patients, but in vitro experiment has not been
reported. So we believe more input is needed for the leptin
expression in AIS, and cytological evidences are warranted to gain
Leptin being a very plausible candidate in AIS, it might be
a very interesting question to begin with whether the alteration of
leptin level is a primary event (i.e. as a result of variations in the
gene) or secondary one (i.e. as an outcome). Association study of
the polymorphisms in Leptin gene promoter did not find significant
differences between cases and controls . However, studies of
polymorphisms in exons and untranslated regions of Leptin gene,
which may as well influence the synthesizing and splicing of leptin,
are lacking. The secretion of leptin is regulated secondary by other
factors. Melatonin, which was widely considered acting a potential
role in the onset and progression of AIS, has several effects in
obesity-related metabolic alterations . The defect of melatonin
may lead to the change of leptin level, resulting in disorder of the
leptin-hypothalamic-sympathetic nervous system, and bringing
about the disease .
The adipogenic ability may affect the leptin level as well. The
adipogenic ability impacts the number and size of adipocytes, and
has great influence on the somatotype [24,25]. There had been
proposed an intrinsic relationship between the adipogenic ability
and BMI . AIS patients had been thought to have
ectomorphic component with lower BMI, characterizing with less
adipose tissue, and expressing less leptin . Thus the adipogenic
ability in AIS may have a relationship with the expression level of
leptin. To evaluate the adipogenic ability, mesenchymal stem cells
(MSCs) as the precursor cells of adipocytes are recommended for
in vitro study [27,28]. Although normal adipogenic ability of MSCs
in AIS patients was reported, the researcher used Oil Red O
staining and quantified with optical densities, which is lack of
calibrator and is inaccurate due to the confounding factor of
various cell numbers . So, more insight in adipogenic
capability of MSCs from AIS is needed.
Additionally, errors in function of leptin have been speculated in
AIS patients . Low serum leptin level stimulates fat
accumulation through central and peripheral pathways and keeps
the body in normal size, but in AIS patients this negative feedback
seems to become invalid. Few studies were conducted to evaluate
the role of leptin receptor. More soluble leptin receptor (sOB-R)
was found in blood samples from AIS girls, and the abnormal
leptin bioavailability was thought to play an important role in the
initiation and progression of AIS . However, the response to
leptin in AIS was not reported. Hence, more details about leptin
receptor and the peripheral response are needed.
Taking all this in consideration and to answer these questions,
we designed a series of genetic and cytological studies. Firstly, we
conducted the detection of single-nucleotide polymorphisms
(SNPs) present in the Leptin gene and did a case-control association
study, in order to examine whether the Leptin gene variations are
associated with genetic susceptibility or curve severity in AIS.
Secondly, we performed adipogenesis assays with bone MSCs, to
evaluate the ability of adipogenesis and to detect the expression of
leptin and leptin receptor in AIS adipocytes. Thirdly, we
performed osteogenesis assays to evaluate the expression of leptin
and leptin receptor in AIS osteoblast. Finally, we performed
factorial designed studies, to examine the responses to leptin
treatment during the adipogenic and osteogenic differentiation of
MSCs. Our result demonstrated that: (1) the polymorphisms of
Leptin gene have no association with genetic susceptibility/severity
of AIS, and the leptin gene expression was normal in AIS induced
adipocytes and osteoblasts after adjustment of the differentiation
rate, indicating the change of serum leptin level probably is
a secondary event; (2) low capability of adipogenesis is found in
AIS girls; (3) the expression of leptin receptor is decreased in AIS
which may lead to (4) hyposensitivity of AIS to leptin. The
abnormal response to leptin may have a role in the pathogenesis of
Genetic Association Study
We performed genetic association study in a systemic fashion.
Screening of exons of the Leptin gene in 45 cases and 45 controls
Reference SNP ID in NCBI
resulted in identification of 19 variations (data not shown). We did
not see obvious accumulation of deleterious rare variations in cases
as compared to controls. We further selected 11 of these variations
with minor allele frequency (MAF) $1% from 45 sequenced
healthy subjects to identify tag SNPs, and conducted an
association study in 446 AIS patients and 550 healthy controls.
LD structure and tagging SNPs. Two LD blocks were
revealed by default settings of haploview, and the frequencies of
the haplotypes in each of them were observed. We identified 6 tag
SNPs named L1 to L6 as shown in Figure 1 and Table 1.
Case-control association study. Age, gender and BMI are
significantly different between the two groups, and therefore are in
need of adjustment (Table 2). The genotype distributions of the 6
SNPs, which were shown in Table 3, were all in HWE. There
were no statistically significant differences of any SNPs between
the patients and controls. The results indicated no obvious
association between any of the tag SNPs and AIS.
Genotype-phenotype studies. We further evaluated if there
is any association of phenotypes with genotypes (Table 4).
Although significant P value (at P,0.05) were observed for the
maximum Cobb angles (MCAs) and different genotypes of
rs11761556 (L6), it appears to be a false positive association as
the average MCAs between three genotypes were almost similar.
As such, no association was observed between the Leptin gene
variations and susceptibility/progression of the disease, suggesting
that the low leptin level is a secondary outcome. Thus we tried to
explore more functional details through cytology experiments, to
figure out whether there is any factor responsible for the low serum
leptin level in AIS patients.
61 Male vs. 385 Female 48 Male vs. 502 Female 0.013*
Table 3. Results of the Case-control association studies.
(n = 446) (n = 550)
127/228/91 166/291/93 0.246
225/188/33 290/222/38 0.492
aThe three values in the genotype column indicate the numbers of
homozygotes(major allele)/heterozygotes/homozygotes(minor allele) in each
bAfter adjusting for age, gender and BMI by logistic regression.
cCalculated for the alleles.
Bonferroni adjustment (All SNPs Padjusted = 1).
Summary of all subjects. Table 5 lists a summary of the
demographic information of MSCs donors. The gender
distribution between two groups was unmatched and the results need to be
discussed based on gender. Although the average ages were similar
in two groups, age as the potential confounding effects should be
taken into account. So we compared the two groups with
adjustment of age. The abnormality of anthropometric parameters
in AIS patients had been widely described in previous researches,
thus the significant differences in weight, BMI and cBMI between
two groups might not be considered as sampling errors [7,9,11].
Given the intrinsic relationship between BMI and the capacity of
adipogenesis, we did not conduct rectifications with BMI .
Identification of MSCs. MSCs were isolated and purified as
previously described. These MSCs were verified morphologically
by a fibroblast-like appearance, and phenotypically by expression
of CD29, CD44, CD105 but not CD34 and CD45 (data not
Comparing the Adipogenic Ability
To assess the expression of leptin in adipocytes and compare the
adipogenic ability of MSCs between two groups, adipogenesis
assay was performed for 12 days. The amount of lipids in induced
adipocytes from AIS group, revealed by Oil Red O staining, was
Average of MCA (Maximum Cobb Angle)a
P value of Kruskal-Wallis Test
aThe three values in the Average of MCA column indicated the mean MCA6Standard Deviation of homozygotes (major allele)/heterozygotes/homozygotes (minor
allele) in each SNP, respectively.
bP,0.05 was considered statistically significant.
cNo sample with homozygotes of minor alleles had been detected in L1, L3 and L4.
less than that from control group. We also quantitively investigated
the expressions of Leptin, Leptin-R, SOCS3 and other adipogenic
markers including PPARc2, LPL and APN.
We compared the genes between the AIS and the control
groups. All of the genes were observed with reduced expression in
AIS group, suggesting the adipogenic ability of MSCs in AIS
patient is lower (see Figure 2 and Table 6). The situations were
a little different when compared the case-control sets based on
their gender. In the female subjects, all genes were reduced
significantly in AIS patients except APN. In the male subjects,
however, only the expressions of Leptin and Leptin-R decreased
significantly. Nevertheless, when used PPARc2 as a reference gene
to adjust the adipogenic differentiation rate, we found that the
Leptin and Leptin-R gene in AIS group decreased insignificantly
in both genders (P.0.05).
Relationship between the genes and clinical
parameters. Correlations between the DCt value of all genes
and the clinical parameters in both genders of AIS patients were
summarized in Table 7.The levels of adipogenic markers (i.e.
PPARc2, LPL and APN) and Leptin showed good consistencies
with each other. Nevertheless, the levels of Leptin-R and SOCS3
generally have no correlation with the adipogenic markers (except
Leptin-R vs. LPL). The Cobb angle did not correlate with the level
of all genes and BMI, suggesting the low adipogenic ability may
not directly impact the severity of the disease.
Leptin receptor expression in induced osteoblast. As
shown in Figure 3, the expression of Leptin, Leptin-R and SOCS3
were significantly decreased in all AIS patients (P,0.001). The
cBMI (kg/m2) 16.3361.86
14 Male vs. 41 Female
18 Male vs. 10 Female
differences remain significant when compared the case-control sets
based on their gender. When we used Runx2 gene as reference to
adjust the different osteogenic rates between two groups, we found
the Leptin-R in AIS still decreased significantly in total and female
data set (P = 0.041 and P = 0.028, respectively), but in male
subjects were similar (P = 0.239). The decrease of Leptin
expression was not statistically significant after the Runx2
adjustment in both genders (P.0.05). No correlation was found
between the level of any gene and Cobb angle (P.0.05).
Immunocytochemistry and western blot of Leptin
receptor. For a single adipocyte, the Leptin receptors seemed
to express at a similar level between AIS and control groups
(Figure 4). However, the leptin receptors were abundant in
undifferentiated MSCs from control sample, but were rare in those
from AIS sample. Besides, the expression of Leptin receptor was
found decreased by immunoblot in AIS induced adipocytes and
osteoblasts (Figure 5).
Responses to Leptin Treatment between AIS and Control
The osteogenesis assay implied that the leptin receptor may be
involved in the progression of AIS. On the other hand, the low
serum leptin in AIS can also be explained as the excessive
sensitivity of the receptor or the signaling pathway, which may in
turn reduce the leptin/leptin receptor levels through a negative
feedback loop. So the effect of leptin in AIS patients was needed to
be studied. Leptin had been reported to restrain adipogenesis and
enhance osteogenesis . We conducted factorial designed
studies to examine the difference of responses to leptin treatment
between AIS and control MSCs.
Effects of leptin in adipogenic differentiation. Oil Red O
staining (Figure 6) showed that the leptin treatment produced
a dose-dependent reduction in the ability of adipogenesis of MSCs
in control group. Specifically, the size and numbers of adipocytes
were significantly decreased after treated with leptin at doses of
0.6 mg/mL. In contrast, leptin treatment brought no change in
AIS group, and all of them displayed adipogenesis levels lower
than in control group.
The relative levels of several genes showed different responsive
patterns between two groups in adipogenic differentiation (Figure 7
and Table 8). Significant Leptin 6 Status interaction existed in the
expression of Leptin, Leptin-R and SOCS3. ANOVA analysis
showed that leptin treatment reduced Leptin, Leptin-R and
SOCS3 expression significantly in control group, but had no
influence on those in AIS group, eliminating the difference
between two groups. Along with the increase of leptin
concentration, the changes of PPARc2, LPL and APN between the two
groups had slight but nonsignificant differences. All the genes in
AIS group sustained in similar levels, implying the induced
adipocytes in AIS group are insensitive to leptin treatment.
Effects of leptin in osteogenic differentiation. Alizarin
Red S staining (Figure 8) showed that leptin dramatically
promoted mineralization of induced osteoblast in control group,
but not in AIS group. The osteogenic ability of MSCs from AIS
group was lower than control when cultured without and with
different doses of leptin.
We also investigated the effects of leptin on expression of several
genes during osteogenesis (Figure 9 and Table 9). Significant
Leptin 6Status interaction were found in the expression of Leptin,
Leptin-R, OCN and OPN. ANOVA results showed that leptin
treatment reduced Leptin, Leptin-R and increased OCN, OPN
significantly in control group. However, leptin did not have any
effect on the expression of these genes in AIS group. Leptin also
appears to promote the expression of SOCS3, Runx2 and ALP in
All P values were calculated with age adjusted.
*P,0.05 was considered statistically significant.
control group, but statistically insignificant. All levels of SOCS3,
OCN, Runx2 and ALP in AIS were significantly lower than
control group, and the leptin treatment enlarged the differences,
indicating a decreased reactivity to leptin in AIS induced
This is the first in vitro study of leptin and leptin receptor in AIS
patients. Our results indicated that Leptin gene variations are not
associated with the susceptibility/progression of the disease, and
the leptin gene expression was normal in AIS induced adipocytes
and osteoblasts after adjustment of the differentiation rate,
implying reduced leptin level is a secondary event. Moreover, in
AIS the adipogenic capability of MSCs is lower, the leptin receptor
level is decreased and the response to leptin is deficient.
Genetic association studies were expected to identify potential
risk variants of AIS and recent GWAS identified some of the
candidate genes [5,6,30]. However, such studies are warranted in
Chinese population. This study confirmed that the genomic
polymorphisms of Leptin gene are not associated with the genetic
susceptibility to AIS, which are consistent with the finding of
Morocz et al . Despite a small P value was observed for the
MCAs and different genotypes of L6, the average MCAs between
the three genotypes were almost similar (25u/26u/30u with wide
standard deviations), suggesting the observation is of no clinical
Previously the adipogenesis levels in AIS group were considered
similar to controls . It was also reported that the osteogenic
ability of MSCs from AIS patients is less, which correlates with
their reduced bone mineral density. We repeated the osteogenesis
assay in our factorial designed study (cultured without leptin) and
found similar results. It has been considered that the adipocyte and
osteoblast lineage pathways of MSCs have an inverse relationship
[31,32]. It is natural to deduce that MSCs from AIS may be prone
to adipogenesis. Surprisingly, our investigations in adipogenic
marker genes indicated that the MSCs from the AIS girls have
a lower capability of adipogenesis. In order to remove any
confounding effect of age distribution in our study, as MSCs from
elder person may tend to undergo adipogenesis , we adjusted
results for age and observed the adipogenic ability was still
significantly lower in AIS group. Our results also showed that the
leptin levels are similar between two groups after adjusting the
adipogenic level, implying that the synthesis and secretion of leptin
are normal in AIS, and the change of leptin level may be
a secondary event. Our study not only explained the thin stature of
AIS girls, but also implied that the low leptin level in AIS girls may
have a relationship with lacking of fat cells. For confirmation, the
relationship between serum leptin level and adipogenic ability
needs further evaluation. Meanwhile, other factors which may
affect the adipogenic ability, such as melatonin signaling, need
investigation so as to figure out the primary event that causes the
abnormal leptin level .
The hyposensitivity of leptin in MSCs from AIS group is elusive
at the moment. We did not observed statistical significance in the
Leptin 6Status interaction of several marker genes, which may be
mainly owing to the limitation of small sample size and great
deviations within the subjects. However, the relative levels of the
genes remarkably demonstrated the low response to leptin of AIS
group. The rare leptin receptor in undifferentiated MSCs as well
as the low level of Leptin-R in osteoblasts, are most likely to be the
direct explanations of the hyposensitivity of leptin. Western blot
also showed decreased leptin receptors in AIS induced adipocytes
and osteoblasts, but these results may be confounded by the low
adipogenesis and osteogenesis levels of AIS MSCs. Besides, the
major leptin signaling pathway, namely JAK2/STAT3 pathway,
may also have a role in the low response to leptin in AIS. The
expression of SOCS3 is induced by the JAK2/STAT3 pathway
and is reported to be an indicator of activity of the signaling
[12,35]. Leptin signaling is also subject to negative feedback
regulation , and leptin treatment may reduce the expression of
leptin and its receptor. Our results showed the expression of
Leptin, Leptin-R and SOCS3 in AIS group were invariant no
matter how much leptin exist. Thus we conclude that the defect of
leptin signaling pathway, if present, may be located in JAK2/
STAT3 pathway. Moreover, there can be another possibility. The
abnormal MSCs are sensitive in none of adipogenesis, osteogenesis
or leptin treatment, in other words, the AIS MSCs have lower
activity. So the hyposensitivity of leptin is probably one of the
behaviors of the inactivity of the MSCs from AIS.
The leptin signaling may contribute to the etiology of AIS.
Leptin is one of the key regulators in pubertal growth and
development. It had been shown to mediate the bone metabolism
and remodeling in vitro and in vivo, in peripheral and in central
nervous system [12,13,17,36,37]. In the leptin receptor-deficient
db/db mouse, where leptin signaling is absent, bone mass and
strength are reduced . Leptin can rescue the skeletal length
and bone mineral density in leptin-deficient ob/ob mouse [39,40].
In AIS patients, an earlier study found the serum leptin level
correlated with bone mineral density, and another study showed
a relationship between free leptin index and curve magnitude in
AIS [19,20]. In this study, we found reduction of Leptin-R level
and hyposensitivity of leptin in AIS. Although there is not enough
evidence to prove the relationship between the hyposensitivity and
the onset of AIS, bipedal db/db mouse model can be constructed
and the role of leptin signaling can be confirmed.
There are some limitations that should be considered in this
study. One of them is the restriction of the small sample size and
the great deviation of subjects, and thus we could not examine the
response to leptin between different genders. The result may be
more convincing if replication is performed. Another weakness is
that the relative level of marker genes may not completely reflect
the differentiation level of MSCs. Oil Red O quantified with
optical densities and alkaline phosphatase activity assay may be
further performed to examine the response to leptin in an overall
In conclusion, our cytological experimental data emphasized
the potential role of leptin signaling in the etiopathogenesis of AIS.
Next step of the studies may be focused on whether and how the
defect of peripheral leptin signaling acts on the body and induces
Materials and Methods
This series of studies were approved by the ethics committee of
Sun Yat-Sen University, and written informed consent was
obtained from all individuals and/or their parents.
Subjects. The study consisted of 446 AIS patients recruited
from the scoliosis clinic. Diagnoses of the patients were confirmed
by experienced surgeons using the Adams forward bending test
and posteroanterior radiographic images of the whole spine. The
inclusion criteria are existing rotational prominence and a
maximum Cobb angle above 10u. Patients with scoliosis secondary to
congenital vertebral malformation, neuromuscular disorders or
syndromic disorders were excluded from the study. Patients
clinical information, including age, gender, curve pattern, Cobb
angle, and Risser sign were recorded. Patients were followed till
skeletal maturity (Risser sign grade 5) unless they accepted surgery.
The minimum follow-up time is one year. If the patient was
treated with bracing, the maximum value in the recorded Cobb
angles was considered as Maximum Cobb Angle (MCA);
otherwise, the Cobb angle obtained at the last visit was MCA. The
maximum Cobb angle ranged from 10u to 140u.
Meanwhile, 550 healthy controls were recruited to the study. All
the controls were examined with the forward bending test to
exclude any scoliosis, and radiographs were taken for validation in
case of any uncertainty. The controls were also eliminated if they
had suffered from any congenital deformity of the spine or had
a family history of scoliosis. All of the cases and controls were Han
Figure 7. Gene expression levels in response to different doses of leptin. The expression levels in control group without leptin treatment
were used as calibrators. Relative expression levels were calculated by using the 22DDCt method.
Leptin 6 Status interaction
Main effect of Status
Main effect of Leptin
Difference between AIS and control
Chinese from south China. Informed consent to DNA analysis was
signed by all subjects or their parents.
Blood samples were collected from each subject through
venipuncture. Genomic DNA was isolated with Tiangen DNA
blood Mini kits (Tiangen, Beijing, China) from peripheral blood
samples collected from each subject.
Resequencing of the Leptin Gene and SNPs
Identification. In order to screen variations in exons of the
Leptin gene, at first resequencing was performed in 45 cases and 45
controls, which were randomly selected from the collection.
Primers were designed for all exons and intron-exon boundaries of
the gene, with 100200 bp extensions into intronic regions (see
Table S1). The PCR products were sequenced in both directions
by ABI Sequence Analyzer 3730XL (Applied Biosystems, Foster
City, CA, USA), and the results were then compared with
sequences retrieved from the UCSC Genome Browser (http://
genome.ucsc.edu/). We identified 19 variations, including 9 novel
rare variations. Data of the novel variations has not been
submitted to GenBank.
Linkage disequilibrium (LD) analysis, haplotype construction
and tag SNP selection of the region were performed with
Haploview 4.2 . SNPs obtained from the 45 sequenced
healthy subjects with minor allele frequency (MAF) $1% were
selected for finding tag SNPs.
Genotyping methods. 6 tag SNPs were identified for further
analyses (see Table 1). They were then detected in 446 AIS
patients and 360 healthy controls by the TaqMan-based
genotyping assay. All of the primers and the probes are listed in
the Table S2. The TaqMan-based genotyping assay was carried
out with the ABI 7500 real-time PCR System (Applied
Biosystems). The reaction mix contained ddH2O 3.25 mL, MgCl2 3 mL,
buffer 1 mL, dNTP 0.25 mL, Primers 0.2 mL, Probes 0.05 mL,
Figure 9. Gene expression levels in response to different doses of leptin. The expression levels in control group without leptin treatment
were used as calibrators. Relative expression levels were calculated by using the 22DDCt method.
ROX reference dye 0.05 mL and DNA template 2 mL, in a total
volume of approximately 10 mL. Amplification was carried out
with a 10 minute cycle at 95uC, 50 cycles at 95uC for 30 seconds
and 63uC for 1 minute. The result was analyzed with the SDS
v1.26System Software (Applied Biosystems). For quality control in
each plate, the sample genotypes confirmed by direct sequencing
were used as positive controls and No Template Control as
Criterion for AIS subjects for cytological experiments were as
follows: (1) diagnosis of AIS was physically and radiographically
confirmed, (2) surgery at our institute, (3) no other skeletal
deformity. Patients complicated with nutritional or metabolic
diseases were excluded. Control subjects were recruited from
patients with fractures. All of the control subjects were confirmed
to have a straight spine by X-ray. Subjects with BMI.23 g/cm2
in both groups were also exclude to avoid overweight or obesity
which may influence the adipogenic ability of MSCs. In total 55
AIS patients and 28 controls were recruited. Bone marrow
samples were provided voluntarily and clinical information was
record. For AIS patients, corrected height was calculated with
Bjures formula (log y = 0.011x 0.177, where y stands for the loss
of trunk height due to the deformity and x stands for maximum
Cobb angle of the primary curve). Corrected BMI (cBMI) is
determined by dividing weight (kg) by the square of the corrected
Isolation and culture of MSCs. MSCs were isolated and
purified by density gradient centrifugation combined with an
attachment culture method . Briefly, mononuclear cells in
bone marrow were separated in a lymphoprep density gradient by
centrifugation at 500 g for 20 minutes, suspended in Dulbeccos
modified Eagles medium (DMEM) with 10% fetal bovine serum
(FBS), seeded and incubated at 37uC/5% CO2. Non-adherent
cells were removed after 48 hr by changing the medium.
Thereafter, the medium was changed every 3 days. When reached
80% confluences, cells were trypsinized (0.25% trypsin) and plated
again. Cells from passages 3,6 were used for the experiments.
Adipogenesis assay. For adipogenic induction, MSCs were
plated at the density of 36104 cells/cm2 in 6-well plated. After
Leptin 6 Status interaction
Main effect of Status
Main effect of Leptin
AIS and control
grown to postconfluence, cells were cultured in an adipogenic
medium for 12 days, which consisted of high-glucose DMEM,
10% FBS, 1 mM dexamethasone, 60 mM indomethacin, 500 mM
3-Isobutyl-1-methylxanthine, 0.01 mg/mL insulin.
Osteogenesis assay. It had been reported that the
osteogenic ability of MSCs from AIS patients is lower than normal .
We conducted the osteogenic differentiation to evaluate the
difference of expression of Leptin, Leptin-R and SOCS3 in
osteoblast between two groups. Osteogenesis assay was performed
on 20 AIS patients versus 17 normal controls (age and gender
matched), which were selected randomly from our MSCs sample
bank described above. Cells were plated and grown until
confluence, then cultured with osteogenic medium for 12 days.
The osteogenic medium contained low-glucose DMEM, 10%FBS,
0.1 mM dexamethasone, 10 mM b-glycerophosphate, and 50 mg/
mL ascorbic acid.
Immunocytochemistry analysis. To examine expressions
of Leptin receptor after adipogenesis, immunocytochemistry was
performed with Hsitostain-Plus kit (ZSGB-BIO, Beijing, China).
After fixed and permeabilized, cells were blocked and incubated
with the antibodies against Leptin receptor (R&D systems,
Minneapolis, MN, USA). Detection was conducted with a DAB
Horseradish Peroxidase Color Development Kit (ZSGB-BIO,
Western blot analysis. Total proteins were collected with
RIPA buffer plus protease inhibitors from cells after adipogenic or
osteogenic induction. Equal amounts (50 mg) of each sample were
resolved on 10.5% SDS-PAGE gel and transferred to PVDF
membranes. After blocked with 5% nonfat dry milk for 1h at room
temperature, membranes were incubated with antibodies against
Leptin receptor (R&D systems, Minneapolis, MN, USA) or
GAPDH (R&D systems, Minneapolis, MN, USA).
Antibodyspecific labeling was revealed by incubation with a
HRPconjugated secondary antibody for 1 h and visualized with the
ECL kit (Millipore, Billerica, MA, USA).
The difference of responses to leptin treatment between
AIS and control MSCs. We also chose 19 AIS patients
randomly and 19 age- and gender-matched normal controls for
the factorial designed study. Cells were treated for 12 days in
adipogenic medium or osteogenic medium without leptin, with
low level leptin (0.015 mg/mL, the average circulating level of
normal adolescent)  or high level leptin (0.6 mg/mL) ,
respectively. Intracellular lipid accumulation in the adipocyte was
indicated with Oil Red O staining. The induced adipocytes was
fixed in 4% paraformaldehyde for 1 hour after washed briefly,
stained with newly filtered Oil Red O for 30 min at room
temperature, then rinsed and photographed. Cells that had
osteogenic differentiated for 21 days was indicated with Alizarin
Red S staining and photographed.
Real-time RT-PCR assay. Total RNA was isolated from
induced adipocyte or osteoblast with RNAiso Plus reagent
(TaKaRa, Dalian, China) and converted to cDNA. Real-time
PCR was performed on a Roche LightCycler 480 System using
SYBR Green Realtime PCR Master Mix (TOYOBO, Osaka,
Japan). The expression of the Leptin gene, Leptin receptor
(LeptinR, long form) gene and Suppressor of Cytokine Signaling 3 gene
(SOCS3, which is induced by the JAK2/STAT3 pathway of leptin
signaling and acts to inhibit the activation of leptin receptor )
were examined. Several adipogenic marker genes were also
investigated, including peroxisome proliferator-activated
receptorgamma 2 (PPARc2), lipoprotein lipase (LPL), adiponectin (APN).
The genes of osteocalcin (OCN), Runt-related transcription factor 2
(Runx2), Alkaline phosphatase (ALP) and osteopontin (OPN) were
chose to evaluate the osteogenic ability. Expression of GAPDH gene
was used as reference. Its noteworthy that the different
differentiation rates between AIS and control in adipogenesis and
osteogenesis may have undesired interferences when comparing the level of
leptin and leptin receptor. So we need reference genes to adjust the
differentiation rates. The PPARc and Runx2 have a crucial role in
adipogenesis and osteogenesis respectively and their level represent
the differentiation level of the MSCs [28,42]. So we use these genes
as reference to calibrate the differentiation rates between AIS and
control. The primer sequences employed are listed in the Table S3.
Each reaction was processed in triplicate and average DCt value
from the whole group was taken. Relative expression levels of each
gene and the calibration of the differentiation rate were obtained by
using the 22DDCt method.
Statistical analyses. In the genetic association study,
HardyWeinberg equilibrium (HWE) test was performed. The allelic
association analyses were performed by Chi-square tests. Logistic
regressions were used to adjust for the confounding effects of age
and gender for genotypic association, while Bonferroni adjustment
was performed for multiple-tests of all SNPs. In
genotypephenotype study, Kruskal-Wallis Test was used for the comparison
of mean maximum Cobb angles among different genotypes in
dataset from cases only, if the Cobb angles showed an abnormal
distribution. In the studies of MSCs, t-test was performed to
compare the DCt value of each gene between AIS and control
group (with the null hypothesis: the Ct differences between target
and reference genes will be the same in AIS vs. control samples),
and the adjustment with age was performed with ANOVA. Test of
Pearsons correlation was used to evaluate the relationship within
the genes, Maximum Cobb Angle, BMI and cBMI. The factorial
designed study was analyzed with ANOVA for factorial designed
data (univariate general linear model), where the Leptin 6 Status
interaction was detected to indicate the differences of responses to
leptin. All analyses were performed using the SPSS software (SPSS
for Windows, Rel. 17.0.0. 2008. Chicago: SPSS Inc.).
We thank our patients and their families for their willing participation. We
thank Professor Yiming Wang (Zhongshan School of Medicine and Center
for Genome Research, Sun Yat-Sen University ) for providing technical
guidance. We also thank Professor Futian Luo (Departmant of Biostatistics
and Epidemiology, Sun Yat-Sen University) for statistical advice.
Conceived and designed the experiments: PS DH. Performed the
experiments: GL WG AL WY LZ. Analyzed the data: YP WG.
Contributed reagents/materials/analysis tools: PS DH. Wrote the paper:
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