Abstract
Purpose
To investigate whether rs11190870 near LBX1 correlates with the susceptibility or curve progression of adolescent idiopathic scoliosis (AIS) in a Han Chinese population.
Methods
A total of 949 AIS patients and 976 age-matched healthy controls were recruited. All the subjects were genotyped using the PCR-based invader assay. Case–control study and case-only study were performed to define the contribution of rs11190870 to predisposition and curve severity of AIS. Additionally, we further conducted a meta-analysis of the study findings together with those of previously reported studies.
Results
A significant association of rs11190870 with AIS was observed in the Han Chinese population (P = 1.8 × 10−9; odd ratio = 1.51; 95 % confidence interval = 1.33–1.71), and AIS patients with TT genotype had a larger Cobb angle than those with TC or CC genotype (P = 0.005). The meta-analysis confirmed that the positive association of this SNP with AIS in the East Asian population.
Conclusions
The SNP rs11190870 near LBX1 is associated with both susceptibility and curve progression of AIS.
Keywords: Adolescent idiopathic scoliosis, Han Chinese, rs11190870, Genetic, Etiology
Introduction
Adolescent idiopathic scoliosis (AIS) is the most common structural spine deformity, with a prevalence of 2–4 % in adolescent, leading to functional disabilities and significant cosmetic problems [1–3]. Despite decades of researches, the specific cause of AIS has not been determined [4]. Many hypotheses have been proposed, including genetic factors, nervous system, skeletal growth, hormones and metabolic dysfunction [5]. It is widely accepted that genetic factors play an important role in the development of AIS [6, 7]. Previous studies have indicated that AIS may be a complex genetic disorder resulting from one or more genetic loci with complex genetic-environment interactions [8–13].
Genome-wide association study (GWAS) is the most robust approach to identify predisposition genes for common diseases and complex traits [14]. Hence, the development of GWAS provides a more comprehensive picture on the possible genes involved in AIS. Recently, Takahashi et al. [15] used a GWAS to compare 1,376 Japanese female AIS patients with 11,297 female controls, and detected a strong association between AIS and a single nucleotide polymorphism (SNP) rs11190870 located at the 3′-flanking region of the LBX1 (ladybird homeobox 1) gene on chromosome 10q24.31. Their association reached genome-wide significance (P = 1.24 × 10−19). The replication study in Hong Kong showed the association between AIS and rs11190870 (P = 9.1 × 10−10) [16]. However, this evidence is limited by its small sample size and little information on the association of rs11190870 with curve severity. Furthermore, Hong Kong Chinese may not be considered equal to Han Chinese of mainland China. Given the history of British colonization and European immigration, Hong Kong Chinese may have more complex genetic background than Han Chinese of mainland China.
To confirm the association between this SNP and AIS, we performed a validation study for the association of rs11190870 with AIS in Han Chinese population residing in Yangtze River region of mainland China. Case–control study and case-only study were conducted to define the contribution of rs11190870 to predisposition and disease severity of AIS.
Materials and methods
Subjects
A total of 1925 subjects were studied. 949 AIS patients (820 girls and 129 boys) with Cobb angle above 20° were recruited from the Spine Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School between May 2005 and August 2011. Curve severity of scoliosis was measured by the Cobb method on the posteroanterior radiograph images of the whole spine. Diagnosis of scoliosis was made for initial Cobb angle ≥10 °. Secondary scoliosis with known etiology was ruled out from the study, including congenital scoliosis, neuromuscular scoliosis, or scoliosis with connective tissue abnormalities. 976 normal control subjects (662 females and 314 males) were age-matched adolescent enrolled from local secondary schools. The control subjects were clinically examined by experienced orthopedic surgeons to rule out any potential spinal deformity. All participants were Han Chinese who lived in and around the Yangtze River region.
The effect of rs11190870 on the curve severity of AIS was determined by a case-only analysis. Since skeletal maturity and bracing can affect natural history of AIS, the patients included in the case-only analysis should be met the following criteria to ensure the intact natural history of AIS: (1) no history of bracing or any other conservative treatment; (2) growth maturation (age >16 years or Risser 5) [17]. Informed consent to DNA analysis was obtained from all subjects and/or their parents. Ethical approval was obtained from the Nanjing University and Drum Tower Hospital Research Ethics Committee.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes using genomic DNA isolation kits (Promega, Madison, WI) according to the manufacturer’s instructions. The primers, probes and reaction conditions were available upon request. SNPs were genotyped by the PCR-based invader assay (Third Wave Technologies) using ABI 7900 (Applied Biosystems, Foster City, CA, WI) [18]. Genotyping was done by laboratory personnel blinded to subject status. Of the samples, 10 % were tested twice to validate the genotyping results with 100 % reproducibility. Two authors independently reviewed the genotyping results, data entry, and statistical analysis.
Statistical analysis
Standard χ2 analysis of contingency tables were used to examine differences of allelic frequencies and genotype distributions between AIS patients and controls. Hardy–Weinberg equilibrium was tested by a goodness-of-fit χ2 test. Odds ratio (OR) and 95 % confidence interval (CI) were calculated using the reported risk allele (T allele) as a reference. One-way ANOVA test was used in the comparison of Cobb angles with different genotypes in the case-only analysis. Meta-analysis was performed with STATA version 11.0 (Stata Corporation, College Station, TX). Statistically significance was considered at P < 0.05.
Results
A total of 1925 subjects (949 cases and 976 controls) were successfully genotyped and subjected to statistical analysis. The distributions of the alleles and genotypes for rs11190870 were presented in Table 1. Genotype frequencies of AIS group and the control group were conformed to the Hardy–Weinberg equilibrium (P = 0.60 and 0.61, respectively).
Table 1.
Allele and genotype frequencies of rs11190870 in adolescent idiopathic scoliosis in a Han Chinese population
| Genotype (%) | P | Allele (%) | P* | OR (95 % CI) | ||||
|---|---|---|---|---|---|---|---|---|
| TT | TC | CC | T | C | ||||
| Total cases | 335 (35.3) | 464 (48.9) | 150 (15.8) | 1134 (59.7) | 764 (40.3) | |||
| Total controls | 236 (24.2) | 496 (50.8) | 244 (25.0) | <0.001 | 968 (49.6) | 984 (50.4) | 1.8 × 10−9 | 1.51 (1.33–1.71) |
| Female cases | 307 (37.4) | 399 (48.7) | 114 (13.9) | 1013 (61.8) | 627 (38.2) | |||
| Female controls | 156 (23.6) | 321 (48.5) | 185 (27.9) | <0.001 | 633 (47.8) | 691 (52.2) | 2.1 × 10−13 | 1.76 (1.52–2.04) |
OR odds ratio, CI confidence interval
* The Cochran–Armitage test
Case–control association study
In accordance with the GWAS and replication study reported previously [15, 16], the risk allele (T allele) lead to a higher risk for AIS in the Han Chinese population (Table 1). T allele frequency of rs11190870 was significantly different between total cases and total controls (P = 1.8 × 10−9) as well as female cases and female controls (P = 2.1 × 10−13).
Case-only association study
A subgroup of 314 skeletally mature AIS patients receiving no bracing or any other conservative treatment previously were analyzed to define the contribution of rs11190870 on curve severity. In general, AIS patients with TT genotype showed significantly higher Cobb angle than the patients with TC and CC genotype (P = 0.005, Table 2). Female AIS patients (n = 258) were isolated alone for one-way ANOVA test, validating a significantly higher Cobb angle in TT genotype compared with TC and CC genotype (P = 0.012, Table 2).
Table 2.
Association between curve severity and rs11190870 in adolescent idiopathic scoliosis
| Population | Genotype | Number | Maximum Cobb angle [mean ± SD] | P |
|---|---|---|---|---|
| Total cases | TT | 110 | 34.1 ± 11.6 | 0.175a |
| TC | 151 | 32.0 ± 13.8 | 0.018b | |
| CC | 53 | 27.2 ± 9.4 | 0.001c | |
| 314 | 0.005d | |||
| Female cases | TT | 87 | 34.7 ± 11.9 | 0.137a |
| TC | 129 | 32.2 ± 14.4 | 0.035b | |
| CC | 42 | 27.3 ± 9.9 | 0.003c | |
| 258 | 0.012d |
aThe difference among TT and TC compared using LSD test
bThe difference among TC and CC compared using LSD test
cThe difference among TT and CC compared using LSD test
dThe difference among TT, TC, and CC compared using one-way ANOVA test
Meta-analysis
We performed meta-analysis between the previous studies [15, 16] and our study. The results showed a significant association between rs11190870 and AIS in East Asian populations. (all P < 0.001, Table 3).
Table 3.
Meta-analysis of the association between rs11190870 and AIS in various populations
| Population | Test for allele frequency of the risk allele (T allele)a | ||
|---|---|---|---|
| P value | OR | 95 % CI | |
| Chinese femaleb | <0.001 | 1.80 | 1.59–2.03 |
| East Asian femalec | <0.001 | 1.64 | 1.53–1.76 |
| Chinesed | <0.001 | 1.60 | 1.44–1.79 |
| East Asiane | <0.001 | 1.58 | 1.48–1.69 |
OR odds ratio, CI confidence interval
aMantel–Haenszel meta-analysis
bHongkongese female and Han Chinese female
cHongkongese female, Han Chinese female and Japanese female
dHongkongese and Han Chinese
eHongkongese, Han Chinese and Japanese
Discussion
GWAS is a powerful method for the detecting genetic contributions to polygenic diseases, and has been increasingly used to study genetic predisposition in AIS that is regarded as one of the most common complex genetic disorders of the musculoskeletal system [19]. However, this method may produce spurious association [20, 21]. Therefore, replications of the associations in different ethnic groups and studies with large sample sizes are important to confirm the results of GWAS [22].
Recently, a GWAS has identified a positive association of a SNP rs11190870 near LBX1 with AIS susceptibility in Japanese [15]. This association has been replicated in the Hong Kong Chinese population [23]. However, Hong Kong Chinese is regarded as a special ethnic group in China. Hong Kong is located to the extreme south of China. Historically, the inhabitants of Guangdong, ancestor of Hong Kong Chinese, have remained rather separate from other parts of China, being spared significant traffic from the north and west by mountain ranges and rivers [24, 25]. Because of the British Colony since the middle of the 19th century, Hong Kong has a long history of European immigration and a high degree of ethnic admixture. As a result, Hong Kong Chinese would be more genetically heterogeneous than Han Chinese of mainland China. Furthermore, a relatively small sample was recruited in the Hong Kong’s study, which may be limited to distinguish false-positives from real associations and conduct a subgroup analysis. Hence, validation of the associations in a large sample size of Han Chinese AIS patients was necessary.
In this study, we identified that rs11190870 near LBX1 was associated with both susceptibility and disease severity of AIS. The total number of AIS patients of the current study was more than three times of that of previous replication study [16], which provided more powerful evidence to support that rs11190870 may account for disease predisposition of AIS. Our study confirmed the earlier finding of positive association in Japanese and Hong Kong Chinese [15, 16]. Furthermore, the results of meta-analysis implicated that the effect of rs11190870 polymorphism near LBX1 would be present across East Asians.
The effect of rs11190870 on AIS severity was also analyzed in a subgroup of skeletally mature AIS. The possible role of rs11190870 in disease progression was assessed quantitatively with the Cobb angle. In the case-only study, rs11190870 was associated with curve severity. AIS patients with TT genotype had a larger Cobb angle than those with TC or CC genotype, indicating that the T allele had an effect on curve development. This was consistent with the findings that T allele was considered as the same risk allele in previous GWAS and replication study [15, 16]. It suggested that rs11190870 might be involved in initiation and progression of AIS.
LBX1 is a homeobox gene and plays an important role in developmental processes. This gene encodes ladybird homeobox 1 and expressed in the central nervous system and skeletal muscle [26–28]. Among the hypothesis of AIS etiology, abnormalities in the central nervous system have long been thought a key AIS etiology [29, 30]. Disturbance of central nervous system may impair somatosensory function and motor adaptation which lead to asymmetry of neuromuscular condition causing AIS [30, 31]. Correspondingly, functional studies in AIS subjects have shown abnormalities in the postural balance and somatosensory function [32–35]. Thus, it remains plausible that LBX1 may be involved in the etiology of scoliosis through somatosensory dysfunction [15]. The SNP rs11190870 is located in the 3′-flanking region of LBX1 on chromosome 10q24.31. As its proximity to gene loci, this SNP is supposed to have an impact on gene regulation; however, the precise role of rs11190870 is still unknown. Functional analysis of the LBX1 and other genes in the 10q24.31 locus might help to understand the real genetic effect on the etiopathogenesis of AIS.
Conclusions
This study suggested that rs11190870 polymorphism was associated with the susceptibility and curve progression of AIS in a Han Chinese population. The results indicated that rs11190870 polymorphism near LBX1 might be both risk factor and disease modifier in the pathogenesis of AIS.
Acknowledgments
This work was supported by the Natural Science Foundation of China (30901570) (81171767). The Manuscript submitted does not contain information about medical device(s)/drug(s). No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.
Conflicts of interest
None.
Footnotes
H. Jiang, X. Qiu and J. Dai contributed equally to this work.
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