Introduction {#s1}
============

Idiopathic scoliosis (IS) may occur and progress at any time during childhood and adolescence [@pone.0076806-Negrini1]. It is defined as a three-dimensional spine deformity, which consists of side curve, deviation of sagittal spinal profile and vertebrae rotation in the transverse plane [@pone.0076806-Negrini1]. According to the Scoliosis Research Society, the angle of lateral curvature measured by Cobb\`s method on standing antero-posterior radiograph should exceed 10° to make the diagnosis justified [@pone.0076806-Cobb1], [@pone.0076806-Stokes1]. Although IS prevalence is reported to be 2--3% in adolescent population [@pone.0076806-Weinstein1], only 10% of the diagnosed children require conservative treatment with corrective bracing and approximately 0.1--0.3% require surgical correction of spinal deformity [@pone.0076806-Negrini1]. The risk of curve progression into severe forms is present until the growth of the spine is completed [@pone.0076806-Weinstein1]. Small angle scoliosis progresses more often in girls than in boys [@pone.0076806-Weinstein1]. The ability to differentiate between non-progressive and progressive IS is crucial for positive outcomes of early intervention [@pone.0076806-Lonstein1].

There exist numerous theories of IS etiology: according to one of the most accepted views, idiopathic scoliosis is a multifactorial disease with an important genetic background [@pone.0076806-Lowe1]. The theory is supported by a higher prevalence of IS in families than in the general population. The occurrence rate was reported at 11.0%, 2.4% and 1.4% for the first, the second and the third degree relatives, respectively [@pone.0076806-WynneDavies1]. Higher concordance was reported for twins: 73% for monozygotic and 36% for dizygotic twins [@pone.0076806-Riseborough1]. Family linkage studies suggested 18 different loci, and over 30 candidate genes were studied for association with IS [@pone.0076806-Gorman1]. Unfortunately, the initial positive results could not be confirmed in the majority of replication studies [@pone.0076806-Gorman1].

Estrogens, being steroid puberty hormones in girls, can act via the Estrogen Receptor type 1 or 2 (ESR1, ESR2) [@pone.0076806-Heldring1] and were suggested to play some role in the pathogenesis of IS [@pone.0076806-Letellier1], [@pone.0076806-Leboeuf1]. There are two *ESR1* single nucleotide polymorphisms (SNPs), described to reveal a certain association with IS. SNP, rs9340799 A/G, often described as XbaI site polymorphism, has been investigated in the Japanese and the Chinese population.

In 2002, Inoue at al. found association of *ESR1* XbaI site polymorphism with (1) curve severity, (2) the risk of progression of \>5° from the initial angle and (3) operation risk in IS Japanese patients [@pone.0076806-Inoue1]. However, in 2011, Takahashi et al. did not confirm the association of this SNP, either with occurrence of IS or curve severity in the Japanese population [@pone.0076806-Takahashi1]. XbaI XX (GG) genotype was described in the Chinese Han population by Wu et al. to be associated with occurrence of IS and to be overrepresented in patients with Cobb angle\>40° and taller than 160 cm [@pone.0076806-Wu1]. However, Tang et al. demonstrated no association of XbaI polymorphism, either with occurrence, or with Cobb angle, nor with anthropometric measurements in the Chinese Han population [@pone.0076806-Tang1]. In 2011, Xu et al. performed a study in the Chinese population and suggested that the X (G) allele at the rs9340799 might be a potential genetic marker, predicting the outcome of brace treatment for IS [@pone.0076806-Xu1].

The rs2234693 C/T is another SNP of *ESR1*, described as PvuII site polymorphism. Zhao et al. postulated association of PvuII with double curve severe scoliosis in the Chinese population [@pone.0076806-Zhao1]. In three other studies the Chinese or the Japanese population, the PvuII site polymorphism alone did not show any association with IS [@pone.0076806-Inoue1], [@pone.0076806-Wu1], [@pone.0076806-Tang1]. Such an association was postulated by Wu et al. in combination with XbaI SNP, therefore, the PpXX (CTGG) might be associated with IS as a "risk" genotype and ppxx (TTAA) as a "protective" genotype [@pone.0076806-Wu1]. The XbaI and PvuII SNPs are situated 46 bp apart and are in strong linkage disequilibrium [@pone.0076806-Becherini1]. These SNPs can form four different haplotype.

The associations of the XbaI or PvuII site polymorphism with occurrence of IS or curve progression or surgery rate have never been investigated in the Caucasian population.

Purpose {#s1a}
-------

The aim of this study was to investigate associations of the two *ESR1* SNPs: XbaI and PvuII, with either the occurrence or progression of IS in Caucasian female patients.

Methods {#s2}
=======

Ethics Statement {#s2a}
----------------

The study protocol was approved by the Institutional Review Board of the Poznan University of Medical Sciences (No 87/09). All the involved patients -- or their parents in case of children -- and all volunteers provided written informed consent.

Patients {#s2b}
--------

From March 2010 till October 2012 two hundred eighty seven Caucasian females with IS, all from one Central European country (Poland), underwent clinical, radiological and genetic examinations. In each case, the age of onset of IS, the age of menarche and treatment history were established. Radiological examination revealed curve pattern, Cobb angle and Risser sign. The Cobb angle under consideration was either the final Cobb angle, defined as the curve angle in skeletally mature patients (N = 253) or Cobb angle, measured in the last follow up of those patients who were skeletally immature (N = 34). Maturity indicators included: (1) the age of at least 16 years, (2) more than 2 years after menarche, (3) Risser sign of 4 or 5 and (4) the end of growth process, defined as height increase of less than 1 cm during previous year. [Table 1](#pone-0076806-t001){ref-type="table"} contains summary of this data.

10.1371/journal.pone.0076806.t001

###### Phenotype description for case-control study.

![](pone.0076806.t001){#pone-0076806-t001-1}

                                                                          Cases                          Controls
  --------------------------------------------------------- --------------------------------- -------------------------------
  females/males                                                          females                          females
  age range (years)                                                      10--59                           16--64
  age mean ±SD (years)                                                  17.9±8.6                          28.3±10
  menarche                                                              yes or no              yes (more than 2 years after)
  family history of scoliosis                                     positive or negative                   negative
  back deformity                                             idiopathic scoliosis (rib hump)           no deformity
  curve pattern                                                            all                              NA
  Cobb angle at final visit                                             10°--114°                           NA
  Cobb angle at final visit mean ±SD                                   39.4°±19.0°                          NA
  angle of trunk rotation in degrees (Bunell scoliometer)                  NA                              \<4°

NA -- non applicable.

Controls {#s2c}
--------

The control group consisted of 182 healthy Caucasian females. The inclusion criteria for the control group were: (1) the age at least 16 years, (2) more than 2 years after menarche, (3) the angle of trunk rotation of less than 4° on examination with scoliometer, (4) the end of growth process, defined as height increase of less than 1 cm during previous year and (5) negative family history of idiopathic scoliosis. Radiological examination was not performed in the control group. [Table 1](#pone-0076806-t001){ref-type="table"} contains summary of this data.

Patients subgrouping {#s2d}
--------------------

Two separate analyses were performed. First, in a case-control study, an association of SNPs with the occurrence of IS was established for all 287 patients and 182 controls. Second, in a case study of skeletally mature patients, an association of SNPs was established with: (1) the scoliosis progression rate, (2) the mean maximum Cobb angle, (3) moderate (\<40°) versus important (≥40°) scoliosis curvature, (4) scoliosis with documented progression of ≥5° Cobb angle versus scoliosis with documented lack of progression, (5) the surgery rate, (6) thoracic versus lumbar scoliosis level and (7) single versus double scoliosis pattern.

For the purpose of analysis of the scoliosis progression rate, the patients were divided into three subgroups, according to the curve progression rate: Non-Progressive IS (NP-IS) with final Cobb angle below 30° (N = 93), Slowly Progressive IS (SP-IS) with the final Cobb angle above 30° but with progression rate below 1°per month (N = 104), Rapidly Progressive IS (RP-IS) with documented radiological progression of ≥1° per month for, at least, 6 months (N = 56). In an analysis of Cobb angle, its maximal values were taken into account. For the purpose of curve size analysis the patients were divided into two subgroups: (1) small or moderate curves with Cobb angle below 40° (N = 161) and (2) important curves with Cobb angle above 40° (N = 92) [@pone.0076806-Wu1]. For the purpose of surgery rate evaluation surgically treated patients (N = 67) were compared to patients after non surgical therapy (N = 186). Regarding the analysis of scoliosis level, the apical vertebra at the thoracic level (Th2-T11) was considered a criterion for thoracic scoliosis while the apical vertebra at the lumbar level (L2-L4) was a criterion for lumbar scoliosis. [Table 2](#pone-0076806-t002){ref-type="table"} contains summary of this data.

10.1371/journal.pone.0076806.t002

###### Phenotype description for case only study in skeletally matured patients with non-progressive (NP-IS), slowly progressive (SP-IS) and rapidly progressive (RP-IS) idiopathic scoliosis.

![](pone.0076806.t002){#pone-0076806-t002-2}

                                                            cases N = 253                                             
  ----------------------------------------------- --------------------------------- --------------------------------- ---------------------------------
  age range (years)                                            16--52                            16--59                            12--54
  Risser                                                       4 or 5                            4 or 5                              all
  skeletal maturity                                              yes                               yes                               no
  back deformity                                   idiopathic scoliosis (rib hump)   idiopathic scoliosis (rib hump)   idiopathic scoliosis (rib hump)
  curve pattern                                                  all                               all                               all
  Cobb angle at final visit (range)                           10°--30°                          30°--65°                          35°--114°
  Cobb angle at final visit (mean ±SD)                       23.3°±5.6°                        40.3°±9.2°                        69.5°±19.2°
  progression rate in degrees per month (range)                  NA                           0.04°--0.95°                       1.0°--3.2°

NA -- non applicable.

Genetic examination {#s2e}
-------------------

### Sample collecting {#s2e1}

2.7 ml of peripheral blood sample from all the patients and from the controls were collected in S-monovette tubes (SARSTEDT AG & Co., Numbrecht, Germany). A total of 469 samples were studied herein. First, a core group of 287 cases of patients with scoliosis was screened for XbaI and PvuII restriction polymorphism. Then, the second group included 182 non-scoliotic phenotype cases, as a control.

### DNA isolation {#s2e2}

The genomic DNA from each patient was obtained from the collected peripheral blood with Axygen AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Scientific, Inc., Union City, CA, USA) according to manufacturer protocol. DNA concentration was determined spectrophotometrically. 50 to 200 ng of gDNA was used for each PCR reaction.

### PCR amplification {#s2e3}

The 1300 bp and 759 bp fragments of the *ESR1* gene were amplified, using primers described in [Table 3](#pone-0076806-t003){ref-type="table"}. The primers were designed using Primer3Plus online software (<http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/primer3plusAbout.cgi>). PCR reactions were carried out in total volume of 25 µl.

10.1371/journal.pone.0076806.t003

###### Primers\' description.

![](pone.0076806.t003){#pone-0076806-t003-3}

                                    Sequence 5′--\>3′           annealing temp   amplicon length
  ------------------------ ----------------------------------- ---------------- -----------------
  ESR1_rs9340799_XbaI_F     CTGCCACCCTATCTGTATCTTTTCCTATTCTCC        71°C            1300 bp
  ESR1_rs9340799_XbaI_R     TCTTTCTCTGCCACCCTGGCGTCGATTATCTGA                   
  ESR1_rs2234693_PuvII_F          AGGCTGGGCTCAAACTACAG               60°C            759 bp
  ESR1_rs2234693_PuvII_R          TCCTTGGCAGATTCCATAGC                          

The reaction mixture for the first *ESR1* gene fragment contained in final concentration: 50 ng of template DNA, 1x KAPA HiFiHotStartReadyMix Polymerase (containing 2.5 mM Mg^2+^; KapaBiosystems, Boston, MA, USA) and 0.3 µM of each primer (Genomed, Gdansk, Poland).

The reaction mixture for the second *ESR1* gene fragment included (final concentration): 200 ng of gDNA, 1x OptiBuffer, 3.5 mM MgCl~2~ solution, 300 nM of each primer, 400 nM of each dNTP, 0,5% DMSO and 1U of BIO-X-ACT DNA polymerase. Compounds used to amplify the second *ESR1* DNA fragment were obtained from Bioline GmbH, Germany.

The thermal profiles for each fragment were polymerase\'s and primer\'s depended and they are shown in [Table 4](#pone-0076806-t004){ref-type="table"}.

10.1371/journal.pone.0076806.t004

###### PCRs reactions thermal profiles.

![](pone.0076806.t004){#pone-0076806-t004-4}

  Step                    ESR1 -- first amplicon   ESR1 -- second amplicon                   
  ---------------------- ------------------------ ------------------------- ---------------- ----
  Initial denaturation         95°C, 5 min                    1               95°C, 5 min     1
  Denaturation                 95°C, 20 sec                  30               95°C, 30 sec    40
  Annealing                    71°C, 15 sec                  30               60°C, 30 sec    40
  Elongation                   72°C, 15 sec                  30              70°C, 1.20 sec   40
  Final elongation             72°C, 3 min                    1               70°C, 5 min     1

The 2 µl of the PCR reaction products were electrophoresed on 2% agarose gel (FMC BioProducts, Rockland, ME, USA) in the presence of ethidium bromide to confirm its identity with molecular mass marker. For 10% of the randomly selected samples half of the reaction mixture were purified according to AxyPrepPCR Clean-up Kit manufacturer\'s protocol and sequenced to confirm its identity to the sequences deposited in NCBI gene sequences database. All experiments were replicated twice.

### Restriction Fragments Length Polymorphism (RFLP) analysis {#s2e4}

The 1.5 µl of each PCR reaction was used for *XbaI* and *PvuII* restriction analysis. SNPs polymorphisms for each fragment were described in [Table 5](#pone-0076806-t005){ref-type="table"}.

10.1371/journal.pone.0076806.t005

###### RFLP SNPs fragment description.

![](pone.0076806.t005){#pone-0076806-t005-5}

                                     recognition site                               Allele                    digestion product length
  ---------------------- ----------------------------------------- ----------------------------------------- --------------------------
  ESR1_rs9340799_XbaI     T[\*](#nt103){ref-type="table-fn"}CTAGA   T[\*](#nt103){ref-type="table-fn"}CTAGA         910bp +390bp
                                                                                    TCTGGA                             1300bp
  ESR1_rs2234693_PuvII    CAG[\*](#nt103){ref-type="table-fn"}CTG   CAG[\*](#nt103){ref-type="table-fn"}CTG        271bp +488 bp
                                                                                    CAGCCG                             759 bp

digestion site.

The digestion was conducted in total volume of 15 µl mixture contained: 1.5 µl of the PCR product, 1 µl FastDigest Green Buffer, 1FDU of the FastDigestEnyzme (*XbaI* or *PuvII*) and DNase free water. The Restriction reaction was incubated in thermocycler 1 hr at 37°C and followed by thermal inactivation of the restriction enzyme at 85°C, 10 min. Afterwards the 10 µl of the reaction were electrophoresed on 2% agarose gel in the presence of ethidium bromide to establish the restriction\'s allele. The results were described as AA or GG or AG for *XbaI* and CC or TT or CT for heterozygotic allele for *PuvII*.

Statistical evaluation {#s2f}
----------------------

The Chi^2^ test was used to determine the differences in allele, genotype and haplotype distribution for the patients with IS, the controls and all the patients subgroups. All archived genotypes were tested for the Hardy-Weinberg equilibrium with Chi^2^ test. A comparison of the mean Cobb angle among the three patients subgroups was verified with the Kruskal-Wallis test. The calculation was made with GraphPad StatMate 2.0. An analysis of the linkage disequilibrium of two polymorphisms was performed with support of the CobeX web tool [@pone.0076806-Gaunt1]. Haplotype evaluation was done with the WinHAP program ver. 1.0 [@pone.0076806-Xu2]. The calculation of the required sample size, based on genotypes frequencies in Central European population revealed that a sample size of 282 cases and 178 controls had 80% power to detect odds ratio (OR) of 1.68 (previously reported OR for the XbaI polymorphism [@pone.0076806-Wu1]) with a significance level of 0.05 two-tailed. The power calculation for the association study was performed with the G\*Power 3.1.7. For all statistics the P value of 0.05 and CI 95% were considered significant.

Results {#s3}
=======

The genotype distribution for the patients, the controls and all the subsequent groups did not deviate from the Hardy-Weinberg equilibrium.

The XbaI and PvuII site polymorphisms showed strong linkage disequilibrium, D\` = 0.838 and r^2^ = 0.5092 with p\<0.0001.

Case-control study: an association of allele and genotype with predisposition to IS {#s3a}
-----------------------------------------------------------------------------------

No significant differences were found, either in allele or genotype frequency between IS patients and healthy controls ([Table 6](#pone-0076806-t006){ref-type="table"}).

10.1371/journal.pone.0076806.t006

###### Allele and genotype frequency for IS patients and healthy controls.

![](pone.0076806.t006){#pone-0076806-t006-6}

  SNP      IS patients N = 287   HWE   Healthy controls N =  182   HWE    p value [a](#nt104){ref-type="table-fn"} dominant[b](#nt105){ref-type="table-fn"}   p value [a](#nt104){ref-type="table-fn"} recessive[c](#nt106){ref-type="table-fn"}   p value [a](#nt104){ref-type="table-fn"} allele de-pendent   Odds ratio (95% CI)           
  ------- --------------------- ----- --------------------------- ------ ----------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ ------------------------------------------------------------ --------------------- -------- -------------------------
  XbaI          Genotype         AA           33.5% (96)                                                     33.5% (61)                                                                                                                                                                                                                       
                                 AG           49.1% (141)          0.89                                      50.5% (92)                                                                              0.56                                                                    0.7696                                   0.9880          0.3685    0.90 (0.54--1.48)\*\*
                                 GG           17.4% (50)                                                     15.9% (29)                                                                                                                                                                                                                        0.99 (0.67--1.48)\*\*\*
                 Allele           A           58.0% (333)                                                    58.8% (214)                                                                                                                                                                                                                      
                                  G           42.0% (241)                                                    41.2% (150)                                                                                                                                                                                                              0.8671      0.97 (0.74--1.26)
  PvuII         Genotype         TT           26.8% (77)                                                     23.1% (42)                                                                                                                                                                                                                       
                                 TC           50.2% (144)          0.93                                      51.1% (93)                                                                              0.76                                                                    0.5572                                   0.4230          0.6046    1.17 (0.76--179)\*\*
                                 CC           23.0% (66)                                                     25.8% (47)                                                                                                                                                                                                                        1.22 (0.79--1.88)\*\*\*
                 Allele           T           51.9% (298)                                                    48.6% (177)                                                                                                                                                                                                                      
                                  C           48.1% (276)                                                    51.4% (187)                                                                                                                                                                                                              0.3601      0.14 (0.87--1.48)

Chi^2^ test.

Dominant model contrasts: for XbaI XX and Xx vs xx, for PvuII PP and Pp vs pp.

Recessive model contrasts: for XabI XX vs Xx and xx, for PvuII PP vs Pp and pp.

There was no significant difference in haplotype frequency between IS patients and healthy controls ([Table 7](#pone-0076806-t007){ref-type="table"}).

10.1371/journal.pone.0076806.t007

###### Haplotype frequency for IS patients and healthy controls.

![](pone.0076806.t007){#pone-0076806-t007-7}

               Haplotype                                          
  ---------- ------------- ------------ ----------- ------------- --------
  Patients    49.1% (282)   8.9% (51)    2.8% (16)   39.2% (225)  
  Controls    44.8% (163)   14.0% (51)   3.8% (14)   37.4% (136)   0.0645

calculated with WinHAP [@pone.0076806-Xu2].

Chi^2^ test.

Case study: an association of allele and genotype with selected parameters of IS {#s3b}
--------------------------------------------------------------------------------

### Association of genotype with progression rate {#s3b1}

The distribution of allele and genotype in rapidly progressive versus slowly progressive versus non-progressive idiopathic scoliosis did not show statistically significant differences ([Table 8](#pone-0076806-t008){ref-type="table"}).

10.1371/journal.pone.0076806.t008

###### Allele and genotype frequency for the patients with non-progressive (NP-IS), slowly progressive (SP-IS) and rapidly progressive (RP-IS) idiopathic scoliosis (N = 253).

![](pone.0076806.t008){#pone-0076806-t008-8}

  SNP      NP-IS     SP-IS      RP-IS   p value [a](#nt109){ref-type="table-fn"} dominant[b](#nt110){ref-type="table-fn"}   p value [a](#nt109){ref-type="table-fn"} recessive[c](#nt111){ref-type="table-fn"}   p value [a](#nt109){ref-type="table-fn"} allele de-pendent                           
  ------- ------- ------------ ------- ----------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ ------------------------------------------------------------ ------ -------- -------- --------
  XbaI      AA     38.7% (36)                                              28.9% (30)                                                                                                                                                    33.9% (19)                                                   
            AG     41.9% (39)   0.21                                       56.7% (59)                                                                              0.11                                                                  42.9% (24)                           0.32                    
            GG     19.4% (18)                                              14.4% (15)                                                                                                                                                    23.2% (13)                                  0.3629   0.5498   0.2194
  PvuII     TT     24.7% (23)                                              26.9% (28)                                                                                                                                                    30.4% (17)                                                   
            TC     53.8% (50)   0.46                                       52.9% (55)                                                                              0.52                                                                  37.5% (21)                           0.06                    
            CC     21.5% (20)                                              20.2% (21)                                                                                                                                                    32.1% (18)                                  0.2041   0.7546   0.2830

Chi^2^ test.

Dominant model contrasts: for XbaI XX and Xx vs xx, for PvuII PP and Pp vs pp.

Recessive model contrasts: for XabI XX vs Xx and xx, for PvuII PP vs Pp and pp.

There was no significant difference in haplotype frequency among the patients with rapidly progressive, slowly progressive and non-progressive idiopathic scoliosis ([Table 9](#pone-0076806-t009){ref-type="table"}).

10.1371/journal.pone.0076806.t009

###### Haplotype frequency for the patients with non-progressive (NP-IS), slowly progressive (SP-IS) and rapidly progressive (RP-IS) idiopathic scoliosis.

![](pone.0076806.t009){#pone-0076806-t009-9}

            Haplotype                                        
  ------- ------------- ------------ ---------- ------------ --------
  NP-IS    48.9% (91)    10.8% (20)   2.7% (5)   37.6% (70)  
  SP-IS    50.5% (105)   6.7% (14)    2.9% (6)   39.9% (83)   0.8482
  RP-IS    45.5% (51)    9.8% (11)    3.6% (4)   41.1% (46)  

calculated with WinHAP [@pone.0076806-Xu2].

Chi^2^ test.

### Association of genotype with IS severity {#s3b2}

The mean maximum Cobb angle in skeletally mature patients did not significantly differ among genotype (XbaI p = 0.8475, PvuII p = 0.1499) ([Table 10](#pone-0076806-t010){ref-type="table"}). For the patients, divided by Cobb angle into two subgroups: moderate below 40° and important above 40°, no significant difference in genotype distribution was found (XbaI p = 0.7434, PvuII p = 0.1170) ([Table 10](#pone-0076806-t010){ref-type="table"}). There was no significant difference of genotype distribution in scoliosis with documented progression of Cobb angle ≥5° versus scoliosis with documented lack of progression (XbaI p = 0.5178, PvuII p = 0.1800). There was no difference of genotype distribution between surgically treated and non surgically treated patients (XbaI p = 0.8052, PvuII p = 0.1637) ([Table 10](#pone-0076806-t010){ref-type="table"}).

10.1371/journal.pone.0076806.t010

###### Genotype according to Cobb angle, progression or surgical treatment (N = 253).

![](pone.0076806.t010){#pone-0076806-t010-10}

                                    XbaI (rs9340799)    p value     PvuII (rs2234693)                  p value                                                         
  -------------------------------- ------------------ ------------ ------------------- ---------------------------------------- ------------ ------------ ------------ ----------------------------------------
  Number of subjects                       85             122              46                                                        68          126           59      
  Mean Cobb angle (±SD)                38.7±21.2       38.6±18.8        38.1±18.0       0.8475[a](#nt114){ref-type="table-fn"}   40.9±19.7    36.4±19.2    40.4±19.5    0.1499[a](#nt114){ref-type="table-fn"}
  Cobb ange\<40°                       34.1% (55)      49.1% (79)      16.8% (27)                                                24.8% (40)   41.3% (88)   20.5% (33)  
  Cobb ange≥40°                        32.6% (30)      46.7% (43)      20.7% (19)       0.7434[b](#nt115){ref-type="table-fn"}   30.4% (28)   41.3% (38)   28.3% (26)   0.1170[b](#nt115){ref-type="table-fn"}
  Documented progression \>5°          31.1% (41)      48.5% (64)      20.4% (27)                                                27.3% (36)   44.7% (59)   28.0% (37)  
  Documented lack of progression       36.4% (44)      47.9% (58)      15.7% (19)       0.5178[b](#nt115){ref-type="table-fn"}   19.0% (23)   55.4% (67)   25.6% (31)   0.1800[b](#nt115){ref-type="table-fn"}
  Patients undergoing surgery          35.8% (24)      44.8% (30)      19.4% (13)                                                29.9% (20)   40.2% (27)   29.9% (20)  
  Patients without surgery             32.8% (61)      49.5% (92)      17.7% (33)       0.8052[b](#nt115){ref-type="table-fn"}   25.8% (48)   53.2% (99)   21.0% (39)   0.1637[b](#nt115){ref-type="table-fn"}

Kruskal-Wallis test,.

Chi^2^ test.

### Association of genotype with IS curve level and curve pattern {#s3b3}

Neither the vertebral level of the main curve apex -- thoracic versus lumbar (XbaI p = 0.3525, PvuII p = 0.8466), nor the numbers of structural curves -- single versus double (XbaI p = 0.5261, PvuII p = 0.8502) did show any association with genotype ([Table 11](#pone-0076806-t011){ref-type="table"}).

10.1371/journal.pone.0076806.t011

###### Genotype according to curve level and curve pattern (N = 253).

![](pone.0076806.t011){#pone-0076806-t011-11}

                         XbaI (rs9340799)    p value     PvuII (rs2234693)   p value[a](#nt116){ref-type="table-fn"}                                         
  --------------------- ------------------ ------------ ------------------- ----------------------------------------- ------------ ------------ ------------ --------
  Main thoracic curve       33.0% (59)      50.8% (91)      16.2% (29)                                                 27.4% (49)   50.3% (90)   22.3% (40)  
  Main lumbar curve         35.1% (26)      41.9% (31)      23.0% (17)                       0.3525                    25.7% (19)   48.6% (36)   25.7% (19)   0.8466
  Single curve              37.5% (39)      46.2% (48)      16.3% (17)                                                 26.0% (27)   51.9% (54)   22.1% (23)  
  Double curve              30.9% (46)      49.7% (74)      19.4% (29)                       0.5261                    27.5% (41)   48.3% (72)   24.2% (36)   0.8502

Chi^2^ test.

### Association of haplotype combination with IS severity {#s3b4}

There were no associations of patients haplotype distribution with curve severity, progression or surgery rate ([Table 12](#pone-0076806-t012){ref-type="table"}).

10.1371/journal.pone.0076806.t012

###### Haplotype according to Cobb angle, progression or surgical treatment.

![](pone.0076806.t012){#pone-0076806-t012-12}

                                     Haplotype                                          
  -------------------------------- ------------- ------------ ----------- ------------- --------
  Patients with Cobb \<40°          49.4% (159)   9.3% (30)    2.8% (9)    38.5% (124)  
  Patients with Cobb ≥40°           47.8% (88)    8.2% (15)    3.2% (6)    40.8% (75)    0.9248
  Documented progression \>5°       46.2% (122)   9.1% (24)    4.2% (11)   40.5% (107)  
  Documented lack of progression    51.6% (125)   8.7% (21)    1.7% (4)    38.0% (92)    0.2976
  Patients undergoing surgery       47.8% (64)    10.4% (14)   2.2% (3)    39.6% (53)   
  Patients without surgery          49.2% (183)   8.3% (31)    3.2% (12)   39.3% (146)   0.8344

calculated with WinHAP [@pone.0076806-Xu2].

Chi^2^ test.

### Association of haplotype combination with IS curve level and curve pattern {#s3b5}

There were no association of patients haplotype distribution with number of the curves or the vertebral level of the main curve ([Table 13](#pone-0076806-t013){ref-type="table"}).

10.1371/journal.pone.0076806.t013

###### Tabel 13. Haplotype according to curve level and curve pattern.

![](pone.0076806.t013){#pone-0076806-t013-13}

                          Haplotype                                          
  --------------------- ------------- ------------ ----------- ------------- --------
  Main thoracic curve    50.6% (181)   7.8% (28)    2.0% (7)    39.6% (142)  
  Main lumbar curve      44.6% (66)    11.5% (17)   5.4% (8)    38.5% (57)    0.0864
  Single curve           50.9% (106)   9.6% (20)    1.0% (2)    38.5% (80)   
  Double curve           47.3% (141)   8.4% (25)    4.4% (13)   39.9% (119)   0.1455

calculated with WinHAP [@pone.0076806-Xu2].

Chi^2^ test.

Discussion {#s4}
==========

The genotype frequency of XbaI and PvuII in 182 controls was comparable with the data, stored in Hap Map CEU (Utah residents with Northern and Western European ancestry) and with publications concerning Caucasian populations: no statistically significant difference in genotype distribution was found ([Table 14](#pone-0076806-t014){ref-type="table"}).

10.1371/journal.pone.0076806.t014

###### Comparison of genotype frequency in 182 controls in this study versus controls from other studies concerning Caucasian populations.

![](pone.0076806.t014){#pone-0076806-t014-14}

                                                                              N    XbaI (rs9340799)   p value [a](#nt121){ref-type="table-fn"}   PvuII (rs2234693)   p value [a](#nt121){ref-type="table-fn"}                    
  ------------------------------------------------------------------------- ----- ------------------ ------------------------------------------ ------------------- ------------------------------------------ ----- ----- ----- --------
  Hap Map CEU                                                                59           29                             25                              5                            0.0711                    28    58    17    0.1889
  Becherini et al. [@pone.0076806-Becherini1] Italy                          610         213                            280                             117                           0.4664                    177   299   134   0.2437
  Patel et al. [@pone.0076806-Patel1] Canada                                 662         273                            307                             82                            0.1324                    197   333   132   0.1003
  Molvarec et al. [@pone.0076806-Molvarec1] Hungary                          180          60                             93                             27                            0.9637                    45    94    41    0.7761
  Ignaszak-Szczepaniak et al. [@pone.0076806-IgnaszakSzczepaniak1] Poland    163          65                             84                             14                            0.0958                    42    78    43    0.7991
  Stavrou et al. [@pone.0076806-Stavrou1] Greece                             145          54                             56                             35                            0.0599                    41    53    51    0.0296
  Roszkowska-Gancarz et al [@pone.0076806-RoszkowskaGancarz1] Poland         414         157                            194                             63                            0.5852                    118   199   97    0.3823

Chi^2^ test.

In this study, two analyses were consecutively performed. First, an association of XbaI and PvuII site polymorphisms with the occurrence of IS was established for 287 patients and 182 controls. Second, an association with selected clinical or radiological parameters was studied for 253 patients, who achieved skeletal maturity: in those patients, the final Cobb angle could be established at the end of growth process. In contrast to the observation published by Wu et al. [@pone.0076806-Wu1], we did not find any significant difference.

in XbaI genotype distribution between females with IS and healthy controls. No overrepresentation of the G allele (X) or the CTGG haplotype (PpXX) was found in the study group. No association of the ESR1 gene polymorphism could be established with either Cobb angle, progression, surgery, curve pattern or curve number. Our results are consistent with the data, published by Tang et al. [@pone.0076806-Tang1] for the Chinese population and with the publication by Takahashi et al. [@pone.0076806-Takahashi1] for the Japanese population. Moreover, we could not confirm the findings of the case only study by Inoue et al. [@pone.0076806-Inoue1] concerning the association of the SNP with curve severity, the risk of progression or the surgery rate.

Taking into consideration this study sample size as well as previously reported data concerning the SNPs risk, it was found that for the OR = 2.0 the power achieved is 95% and for the reported OR = 1.68 the power is 80% [@pone.0076806-Wu1].

In this study, much attention was paid to precise phenotype evaluation in the patients. The division of natural course scoliosis into NP-IS, SP-IS and RP-IS subgroups was based on clinical relevance, as proposed by Dimeglio [@pone.0076806-Dimeglio1]. Early detection of RP-IS patients is crucial for effective treatment outcome [@pone.0076806-Kotwicki1]. On the other hand, according to Weinstein et al. [@pone.0076806-Weinstein2], scoliosis with Cobb angle below 30° does not affect patient\`s quality of life and remains stable throughout the lifetime without tendency for progression [@pone.0076806-Weinstein2]. This clinical form was named in our study Non-Progressive IS (NP-IS).

Taking into consideration gender shift, demonstrated by a higher progression rate in adolescent females, in association with puberty, sex hormones may have influence on curve progression [@pone.0076806-Weinstein1]. That is why genetic studies in the *ESR1* area are justified. The PvuII and XbaI site polymorphisms are located in intron 1, 46 bp apart, of the ESR1 gene. It is postulated that the SNPs do not exert any direct impact on the receptor structure [@pone.0076806-Kobayashi1], however a regulatory role of the SNPs was suggested, as a potential enhancer of gene expression [@pone.0076806-Hill1]. The XbaI and Pvu II SNPs were described to be associated with the age of menarche. Stavrou et al. described a later onset of menarche in patients with XbaI XX (GG) genotype and with XP (GC) haplotype [@pone.0076806-Stavrou1]. Delayed hormonal maturity may be associated with longer period of risk of progression in IS and bigger final Cobb angle [@pone.0076806-Yim1]. SNPs association with bone mineral density (BMD) was described [@pone.0076806-Kobayashi1], the data being, however, incoherent [@pone.0076806-Ioannidis1]. Low BMD values in IS were indicated in many studies [@pone.0076806-Li1] as a possible linkage between *ESR1* polymorphism and IS. Estrogen receptor is expressed both in osteoblasts and osteoclasts [@pone.0076806-Pensler1], [@pone.0076806-Komm1].

Conclusions {#s5}
===========

In the Central European population, no association was found between *ESR1* XbaI and PvuII site polymorphisms and IS, nor any of the previously reported associations was confirmed, regarding the surgery rate, curve severity or curve progression.

This manuscript does not include any information about medical device (s) or drug (s). No benefits in any form have been or will be received from any commercial entity related directly or indirectly to the subject of this manuscript. We would like to thank all participants for their support in this study.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: TK MA MK. Performed the experiments: PJ TK MA MK. Analyzed the data: PJ TK MK. Contributed reagents/materials/analysis tools: PJ TK MA MK. Wrote the paper: PJ TK MA MK.
