Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in the Chinese Han Population
et al. (2013) Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in
the Chinese Han Population. PLoS ONE 8(1): e53234. doi:10.1371/journal.pone.0053234
Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in the Chinese Han Population
Wenjie Gao 0
Yan Peng 0
Guoyan Liang 0
Anjing Liang 0
Wei Ye 0
Liangming Zhang 0
Swarkar Sharma 0
Peiqiang Su 0
Dongsheng Huang 0
Nicholas John Timpson, University of Bristol, United Kingdom
0 1 Department of Orthopedic Surgery, The First Affiliated Hospital of Sun Yat-sen University , Guangdong , China , 2 Department of Orthopedic Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University , Guangzhou, Guangdong , China , 3 Seay Center for Musculoskeletal Research, Texas Scottish Rite Hospital for Children, Dallas, Texas, United States of America, 4 School of Biology and Chemistry, Shri Mata Vaishno Devi University , Katra , India
Background: Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities found in adolescent populations. Recently, a genome-wide association study (GWAS) in a Japanese population indicated that three single nucleotide polymorphisms (SNPs), rs11190870, rs625039 and rs11598564, all located near the LBX1 gene, may be associated with AIS susceptibility . This study suggests a novel AIS predisposition candidate gene and supports the hypothesis that somatosensory functional disorders could contribute to the pathogenesis of AIS. These findings warrant replication in other populations. Methodology/Principal Findings: First, we conducted a case-control study consisting of 953 Chinese Han individuals from southern China (513 patients and 440 healthy controls), and the three SNPs were all found to be associated with AIS predisposition. The ORs were observed as 1.49 (95% CI 1.23-1.80, P = 5.09E-5), 1.70 (95% CI 1.42-2.04, P = 1.17E-8) and 1.52 (95% CI 1.27-1.83, P = 5.54E-6) for rs625039, rs11190870 and rs11598564, respectively. Second, a case-only study including a subgroup of AIS patients (N = 234) was performed to determine the effects of these variants on the severity of the condition. However, we did not find any association between these variants and the severity of curvature. Conclusion: This study shows that the genetic variants near the LBX1 gene are associated with AIS susceptibility in Chinese Han population. It successfully replicates the results of the GWAS, which was performed in a Japanese population.
Funding: This work was supported by the National Natural Science Foundation of China (No. 81171674 and No. 81071703), Research Fund of Social
Development of Guangdong Province (No. 2010B031900023), Research Fund of Popular Science of Guangzhou City (No. 2011KP012) and the Fundamental
Research Funds for the Central Universities (No. 11ykzd10). 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 structural,
tridimensional spinal deformity characterized by lateral curvature of the
spine with Cobb angle (which is a measurement used for
evaluation of curves in scoliosis ) greater than 10u. It affects
23% of the adolescent populations .
Heritable and genetic factors have been found to play a vital
role in the occurrence and development of AIS [4,5]. Several loci
associated with predisposition to AIS have been identified in
genome-wide linkage studies in such regions as 6p, 10q, 18q,
19p13.3, 17p11, 19p13, 8q12, 9q31.2-q34.2, 17q25.3-qtel, 12p,
and Xq . Single nucleotide polymorphisms (SNPs) in the
genes for estrogen receptor a (ESR1), estrogen receptor b (ESR2),
matrilin 1 (MATN1), melatonin receptor 1B (MTNR1B),
tryptophan hydroxylase 1(TPH1), interleukin-6 (IL-6) and matrix
metalloproteinase-3 (MMP-3) have been reported to be associated
with AIS predisposition . However, so far these studies
have not been replicated in other ethnic groups .
Polymorphisms of ESR1, ESR2, MATN1, insulin-like growth
factor-I (IGF-I), tissue inhibitor of metalloproteinase-2 (TIMP-2),
G protein-coupled estrogen receptor 1 (GPER), and neurotrophin
3 (NTF3) have been reported to be associated with the severity of
curvature in AIS [1517,2630]. These might be the modifier
genes for AIS, but at present there is a lack of conclusive functional
Genetic association studies are the means of identifying risk
variants in complex traits, and replication studies that confirm
their findings in other ethnic groups are quite necessary [4,3133].
Recently, a genome-wide association study (GWAS) was
performed in a Japanese population, and three SNPs (rs11190870,
rs625039 and rs11598564), all of which were located near the gene
LBX1 on chromosome 10q24.31, were reported to be associated
N (observe/bracing/surgery) 513(336/108/69)
Mean age6SDa (years)
Age range (years)
Age at diagnosis (years)
Mean MCAb6SD (u)
MCA range (u)
aStandard deviation (SD).
bThe maximum Cobb angle (MCA).
cNot applicable (NA).
with AIS susceptibility . Two association studies conducted in
Chinese Han population from Hong Kong and Yangtze River
region of mainland China replicated the association between AIS
and rs1190870 [34,35], and rs11598564 was among the top 100
SNPs identified in a GWAS conducted in the United States .
In order to determine whether rs11190870, rs625039, and
rs11598564 are associated with a predisposition to AIS in Chinese
Han population of Southern China, we conducted a case-control
study involving 513 AIS patients and 440 control subjects. A
caseonly study including a subgroup of AIS patients was also
performed to determine the effects of these variants on the
severity of the condition.
The study has been approved by the Ethical Committee of the
First Affiliated Hospital of Sun Yat-sen University and the Ethical
Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen
University. Written informed consent was obtained from all
subjects or their parents in the case of children.
All the individuals who participated in the present study were of
Chinese Han ethnicity and from Guangdong Province in Southern
Five hundred and thirteen AIS patients were collected from the
First Affiliated Hospital and Sun Yat-sen Memorial Hospital of
Sun Yat-sen University. They included 336 patients with mild
scoliosis (who only needed observation), 108 with moderate
scoliosis (treated with bracing), and 69 with severe scoliosis
(treated with surgery). The diagnosis of idiopathic scoliosis is one of
exclusion, and it is made only when other causes of scoliosis have
been ruled out [37,38]. Based on the age of the patient at the time
of his or her first diagnosis of scoliosis, idiopathic scoliosis can be
subdivided into three groups: infantile, before three years of age;
juvenile, between three and ten years of age; and adolescent,
between age ten and skeletal maturity [37,39]. In our study, all
patients provided detailed histories, accepted physical
examinations, underwent standard up-standing posteroanterior
radiography of the whole spine, and other testing (if necessary), such as
MRI, CT, and nuclear scintigraphy etc [37,40]). All patients were
ascertained for diagnosis of AIS at the age of 1016 years by at
least two spinal surgeons. Patients with congenital scoliosis and
scoliosis secondary to neuromuscular disorders, endocrine
disorders, skeletal dysplasia, connective tissue abnormalities, and
syndromic disorders were excluded. The controls typed for the
analysis comprised: 1) 363 young students recruited during
scoliosis screening at middle and primary schools; 2) 77 young
fracture patients selected from the First Affiliated Hospital and
Sun Yat-sen Memorial Hospital of Sun Yat-sen University.
Adams forward bend test and scoliometers were used to screen
individuals for scoliosis [37,38]. (The sensitivities of the Adams
forward bend test and scoliometer have been reported to be
84.37% and 90.62%, respectively, and specificity is 93.44% and
79.76% respectively .) Radiographs were taken for validation
in case of any uncertainty. Routine history-taking and physical
examinations were also conducted to exclude other deformities of
aRisk alleles were indicated in boldface.
the skeletal system, hereditary diseases, and disorders affecting
bone growth and metabolism. At least two orthopedic surgeons
assessed the controls. Only when all surgeons were in agreement
was the participant included in the study.
AIS tends to progress until skeletal maturity, and bracing can
change the natural course of the condition . Only a subgroup
of AIS patients (N = 234), those who reached the endpoints of
curve progression [surgical fusion for AIS or skeletal maturity
(defined as age 16 or Risser sign 5)] and had not undergone
bracing, were included in the case-only study. The severity of
spinal curvature was measured using the Cobb method on
standard up-standing posteroanterior radiography of the whole
spine . The measurement was made by drawing tangents along
the superior endplate of the superior end vertebra and the inferior
endplate of the inferior end vertebra. The Cobb angle was here
defined as either the angle between the tangential lines or the
angle between two lines drawn perpendicular to the tangents (the
two angles are identical) . The maximum Cobb angle (MCA)
of the primary curve was used to assess the severity of AIS. For
patients who underwent surgery, MCA was taken before surgery,
and for those who were under observation, MCA was taken during
the latest follow-up.
After written informed consent was obtained, participants basic
information, such as name, age, sex, ethnic group, and birthplace,
and clinical information, such as Cobb angle and Risser sign
[44,45], were recorded, and blood samples were collected.
Genomic DNA was isolated from 200 ml blood per patient using
Tiangen DNA Blood Mini Kits (Tiangen, Beijing, China)
according to the manufacturers instructions. Genomic DNA was
diluted to a final concentration of 1015 ng/mL for genotyping
MeanSD age (years) for genotype
aP-values were calculated using KruskalWallis test.
assays. Polymorphism-spanning fragments were amplified using
polymerase chain reaction (PCR) and genotyped using the
MassArray system (Sequenom, San Diego, CA, U.S.) with primers
(Table S1) at the Beijing Genomics Institute in Shenzhen, China,
as described previously . For quality control, three
templatefree controls and sixteen duplicated samples were used per
384sample plate, and the results were 100% consistent. Genotyping
was conducted blindly, and the call rate for each SNP was 100%.
The genotype distributions of the SNPs were all in
HardyWeinberg equilibrium (HWE) was evaluated in both the
case and control groups for all SNPs. Differences between cases
and controls with respect to allele frequency were evaluated using
the Chi-Square test, and the Cochran-Armitage trend test was
used for genotype frequency. The allelic odds ratios (ORs) and
their 95% confidence interval (CI) ranges were calculated.
Bonferroni adjustment was performed for multiple-test
corrections. Logistic regression was used to adjust the confounding effects
of age and sex and determine the effect of interaction between
SNP and sex. The KruskalWallis test was used to evaluate the
association between age and genotype. The KruskalWallis test
and ordinary least squared regression were used in the comparison
of maximum Cobb angles (MCAs) among different genotypes.
Linear regression based beta coefficients were also calculated to
reflect the changes in MCAs per risk allele. The power calculation
for the association study was performed with the NCSS/PASS
software (NCSS, Kaysville, UT, USA). Linkage disequilibrium
(LD) and haplotype analyses were performed using Haploview 4.2
. The other statistical tests were performed using SPSS 13.0
(SPSS Inc., Chicago, IL, U.S.) and PLINK 1.07 .
This study included 513 AIS patients with Cobb angles over 15u
and 440 control subjects (Table 1). The distributions of the alleles
and genotypes for the three SNPs are given in Table 2. All the
SNPs were all found to be associated with AIS predisposition, the
ORs were observed as 1.49 (95% CI 1.231.80, P = 5.09E-5), 1.70
(95% CI 1.422.04, P = 1.17E-8) and 1.52 (95% CI 1.271.83,
P = 5.54E-6) for rs625039, rs11190870 and rs11598564,
respectively. And the present study had a power of 91%, 91% and 77%
for rs625039, rs11190870 and rs11598564 respectively to show the
effects reported in the previous GWAS .
The linkage disequilibrium coefficient (r2) between rs625039
and rs11190870 was 0.59, and the coefficient between rs11190870
and rs11598564 was 0.70 (Figure S1). Haplotype analysis did not
show any association stronger than that observed between the
SNP rs11190870 and AIS (Table 3).
We also evaluated possible confounding factors, such as age and
sex using logistic regression, and the adjusted ORs for rs625039,
rs11190870 and rs11598564 were 1.60 (95% CI 1.262.03), 1.65
(95% CI 1.362.01), and 1.51(95% CI 1.241.84), respectively
(Table S2). No interaction effect between SNP and sex on the risk
of AIS was observed (Table S2). Also, no association was found
between age and genotype for the SNPs in either the case or
control group (Table 4). When the population was stratified by sex,
associations between SNPs and AIS predisposition were detected
among female individuals, but not among male individuals.
However, this might be due to the small sample size of male
subset (66 cases and 151 controls) (Table 5).
This study included 234 AIS patients who had reached the
scoliosis curve endpoints [surgical fusion for AIS or skeletal
maturity (defined as age 16 or Risser sign 5)] and had never been
braced. No difference with respect to MCAs was found among
genotypes for any of the SNPs (Table 6). A sensitivity analysis was
also performed on all 234 cases and 440 controls. The SNPs were
still found to be associated with AIS predisposition, and the ORs
showed little difference from the previous case-control study
(Table S3). In this way, in the present study, none of the SNPs
were found to be associated with the severity of spinal curvature.
Mean MCAa SD(6)
b coefficient (Standard error) b
aMaximum Cobb angle (MCA).
bb coefficients, standard errors and P-values were calculated using ordinary least squared regression.
cP-values were calculated using the KruskalWallis test.
In the case-control study, all three genotyped SNPs were found
to be associated with AIS predisposition. We also found G, T, and
G to be risk alleles for rs11598564, rs11190870, and rs625039,
respectively, the same as those reported in the previous Japanese
GWAS . The association between common variants near LBX1
and AIS predisposition found in Japanese population was
successfully replicated in this Chinese Han population.
Studies on animal models showed that damage to the sensory
area of the spinal cord or posterior rhizotomy could cause scoliosis,
indicating that somatosensory dysfunction might play a significant
role in AIS . In clinical studies, it has been noted that
growing children with functional or structural disorders of the
somatosensory pathway are more susceptible to scoliosis than their
healthy counterparts . In AIS patients, the prevalence of
somatosensory disorders is much higher than in the general
population . LBX1 is a homeobox gene expressed in the
dorsal part of the spinal cord and hindbrain. It was first cloned by
Jagla K et al., and it has been reported to act as a selector gene in
the determination of the fates of dorsal spinal and hindbrain
somatosensory neurons . In Lbx12/2 mice, the
morphology and neuronal circuitry of the dorsal horn are aberrant,
suggesting that LBX1 is critical to the development of the sensory
pathway in the spinal cord . Because variants near LBX1 were
found to be associated with predisposition to AIS in the present
and previous studies, it is possible that abnormal LBX1 expression
might contribute to AIS by causing somatosensory function
Replication is essential for substantiation of the positive findings
of association studies and identification of common causes of
disease among different populations. However, this process often
fails in independent studies, including AIS association studies [21
25]. Since Xu S etc.  reported that there are remarkable
genetic differences among Chinese Han populations from different
regions of China, ours and other recent two association studies
[34,35], which were conducted in Chinese Han population from
different regions of China (from Hong Kong, Yangtze River
region and Southern region of China respectively), all replicated
the association between AIS and rs1190870, and strongly support
that rs11190870 may account for disease predisposition of AIS in
Chinese Han population. Yet further replication studies must be
performed in other ethnic groups, since of the SNPs evaluated
here, only rs11598564 was among the top 100 SNPs identified in a
recent large-scale GWAS conducted in the United States . It is
also important to note that these three SNPs are all located in the
flanking region of LBX1 (rs11190870 and rs11598564 are in the 39
region, and rs625039 is in the 59 region). The potential functions
of these SNPs are still unclear. These variants may act as
regulatory elements for LBX1, affecting the quality and quantity of
LBXI mRNA [63,64]. Then we searched these SNPs in the
recently published ENCODE database (http://www.regulomedb.
org), and observed rs625039 may minimally affect binding of motif
Pax-4; rs11190870 may minimally affect binding of 9 motifs
(Lhx5, Pbx-1b, Oct-4, Oct-1, Pou2f2, Lhx3, Pou2f3, Arid3a and
Octamer), whereas, rs11598564 may not cause any motif change.
It is also possible that these are just markers of LD, associated with
actual disease-causing variants. Functional analysis of these
variants and targeted resequencing of the whole LD block must
be performed to identify functional variants. This may shed
further light on the mechanisms underlying the pathogenesis of
Adjusting the association for age and sex effects using a logistic
regression approach did not substantially change the nature of our
findings. The adjusted ORs showed little difference from the
preadjustment values (Table S2). No association was found between
age and genotype for any SNP, indicating that age has no effect on
the distribution of the genotypes evaluated in the present study.
Although the control group included subjects who were young and
had immature skeletal systems [(,10 years) 7; ($10 and ,14
years) 135; ($14 and ,16 years) 223; (.16 years) 75] at the time
of sample collection, some of them might go on to develop the
disease over time. However, in light of a previous epidemiological
studies, only a very small number of them (,1%) would develop
clinically significant AIS .
Disease modifier genes might be useful for predicting the
progression of disease and helpful in early clinical investigation
and treatment . For this reason, we conducted a case-only study
including a subgroup of AIS patients. Because AIS can progress
until skeletal maturity and bracing can change the natural course
of AIS, only patients who had reached the endpoints of curve
progression without ever having been braced were included .
In the present study, we found none of these three SNPs to be
associated with the severity of spinal curvature in AIS, which
indicates that LBX1 might not be a disease modifier gene for AIS.
However, further study with larger sample size and conclusive
functional study are needed to confirm this.
This study has some limitations that should be addressed. First,
a significant difference was observed between male and female
individuals with respect to prevalence of AIS. The ratio of
femaleto-male prevalence was found to be 3.6:1 . When we stratified
the sample by sex, little evidence for association was found among
males (Table 5). Because the variants were found to have the same
effects on AIS predisposition in female individuals from both
Chinese and Japanese populations, and no interaction effect
between SNP and sex was observed (Table S2), we conclude that a
lack of power [due to the small size of the male sample population
(66 cases and151 controls)], rather than the sex specific effects of
the variants in Chinese Han population, is the most plausible
explanation for this lack of association. Further study with larger
sample size is needed. Second, AIS is a complex trait, and certain
non-genetic risk factors for AIS, such as oteopenia and late
menarche have been reported [4,65,66]. However, these data
were not collected in the present study, which limited our ability to
evaluate of gene-environment interactions.
Table S1 Primer sequences used for genotyping the
SNPs with the Sequenom platform.
Table S2 Adjustment for age and sex and investigation
of the interaction effect between SNP and sex using
Table S3 Case-control study with 234 cases in whom the
severity of the curve was measurable and 440 controls.
We would like to thank all participants for their support in this study. We
also thank Dr. Yong Huang of the Department of Biostatistics and
Epidemiology, Sun Yat-Sen University for statistical advice.
Conceived and designed the experiments: DH PS. Performed the
experiments: WG YP. Analyzed the data: WG YP GL. Contributed
reagents/materials/analysis tools: PS DH AL WY LZ. Wrote the paper:
WG YP SS.
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