ASSOCIATION BETWEEN ESR1 COMMON GENETIC POLYMORPHISMS AND CURVE SEVERITY OF IDIOPATHIC SCOLIOSIS IN BULGARIAN PATIENTS: A CASE-CONTROL STUDY

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1 Доклади на Българската академия на науките Comptes rendus de l Académie bulgare des Sciences Tome 68, No 6, 2015 MEDECINE Génétique médicale ASSOCIATION BETWEEN ESR1 COMMON GENETIC POLYMORPHISMS AND CURVE SEVERITY OF IDIOPATHIC SCOLIOSIS IN BULGARIAN PATIENTS: A CASE-CONTROL STUDY Svetla T. Nikolova, Vasil Ts. Yablanski, Evgeni N. Vlaev, Luben D. Stokov, Ivo M. Kremensky, Alexey S. Savov (Submitted by Corresponding Member D. Toncheva on March 26, 2015) Abstract A case-control study was performed on 80 patients with idiopathic scoliosis (IS) and 160 healthy controls from Bulgarian population. Its objective was to investigate the association between common genetic polymorphisms of ESR1 and progression of IS among Bulgarian patients. The current consensus on IS maintains that it has a multifactorial etiology with genetic predisposing and modifying factors. Molecular detection of the polymorphisms of ESR1 was performed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). The statistical analysis was performed by chi-squared test. This case-control study revealed statistically significant association between the PvuII polymorphism (rs ) and susceptibility to IS and curve severity (p < 0.05). XbaI polymorphism (rs ) was not found to be correlated with the onset or progression of IS (p > 0.05). ESR1 gene could be considered as susceptibility and modifying factor of IS. The identification of molecular markers with diagnostic and prognostic value could facilitate the therapy decisions and permit early stage treatment of the patient with the least invasive procedures. An extended population-based case control study is necessary to confirm the contribution of these polymorphic variants to the development and progression of IS in Bulgarian population. Key words: idiopathic scoliosis, ESR1, curve severity This work was supported by Medical University-Sofia (Grant No 49/2012.) 783

2 Introduction. The current consensus on IS maintains that it has a multifactorial etiology with genetic predisposing and modifying factors [ 1 ]. The estrogen receptor 1 gene (ESR1) was shown to be expressed in both human osteoblasts and osteoclasts [ 2 ] and mutations of the ESR1 gene were shown to cause bone loss and delayed skeletal growth in affected humans [ 3 ]. The specific XbaI (rs ) and PvuII (rs ) polymorphisms of the ESR1 gene were associated with low bone mineral density (bmd) in a Bulgarian female population sample [ 4 ]. A study conducted by Inoue et al. [ 5 ] in 2002 was the first one which investigated the role of the ESR1 XbaI and PvuII polymorphisms in the pathogenesis of IS among Japanese patients and concluded that the ESR1 XbaI polymorphism constitutes important factor for the curve progression. No association was found between the ESR1 restriction polymorphisms and IS in Polish females [ 6, 7 ]. In the present study the association of the single nucleotide polymorphisms (SNPs) XbaI and PvuII of ESR1 with IS was investigated in Bulgarian patients. All participants in the study were informed about its purpose and were included only after the subjects/families signed their informed consent. Materials and methods. Patients. Patients with IS (n = 80) were recruited with the help of orthopaedic surgeons from Tokuda Hospital Sofia and University Orthopedic Hospital Prof. Boycho Boychev. The IS diagnosis was confirmed clinically and radiologically. The curves were measured by the Cobb method. The mean value of Cobb angles was 54.6 ± The mean age at the beginning of the disease was 11.2 ± 3.1 years. In this study, male (n = 15) and female (n = 65) patients were included. Controls. The control group (n = 160) including healthy subjects without clinical signs of IS was recruited from a pool of unrelated gender-matched volunteers from other Units and Clinics of Tokuda Hospital Sofia, National Genetic Laboratory, hospital staff members and students. The controls were selected among adult patients with skeletal maturity with negative family history of IS. Radiological examination was not performed in the control group. Genotyping. Genomic DNA was extracted from the peripheral blood leucocytes using magnetic bead technology (chemagic DNA Blood Kit special, Chemagen). The polymorphic region of the ESR1 was amplified by polymerase chain reaction (PCR) with a previously published primer set [ 5, 6 ]. The PCR was carried out in a reaction mix of 20 µl containing 100-ng DNA and 10X Prime Taq buffer (Genet Bio), 10 mm dntps Mixture (Genet Bio), 20 pmol Forward and Reverse primers (AlphaDNA), and 0.1 U Prime Taq DNA Polymerase (Genet Bio). PCR ampifications were performed in an AB 2720 Thermocycler (Life Technologies) with an initial denaturation at 94 C for ten minutes and a final extension of ten minutes at 72 C. The following thermal cycle was repeated 30 times: denaturation at 94 C for 60 seconds, annealing for 60 sec at 59 C, and extension at 72 C for 120 sec. 784 S. Nikolova, V. Yablanski, E. Vlaev et al.

3 The PCR products were cleaved with the appropriate restriction enzymes (New England Biolabs), according to the manufacturer s instructions, and the digested products were separated on 3% agarose gel in VG-SYS Horizontal Electrophoresis System (Biochrom). The restriction enzymes and the lengths of the fragments representing the genotypes of the genes are presented in Table 1. Statistical analysis. The clinical characteristics, Cobb angle, and ESR1 genotypes were compared using χ2 test with a P value of 0.05 as statistically significant. All calculations were performed with the statistical program SPSS 19.0 software package for Windows. Results. The overall frequency of the PP genotype of the ESR1 PvuII polymorphism in the patients with IS was two times higher than in the controls (p = 0.006) and the frequency of the P allele of the ESR1 PvuII polymorphism in the patients with IS was also higher than in the controls (p = 0.045). In conclusion, the homozygous PP genotype of ESR1 was associated with a higher risk of scoliosis (OR = 2.37; CI: ) and the presence of the P allele alone could be considered as susceptibility factor to IS. The overall frequency of the XX genotype of the ESR1 XbaI polymorphism in the patients with IS was higher than in the controls (p = 0.09) and the frequency of the X allele of the ESR1 XbaI polymorphism in the patients with IS was also higher than in the controls (p = 0.12) but no significant association was detected (p > 0.05). In conclusion, the presence of the X allele alone and the homozygous XX genotype of ESR1 could not be considered as susceptibility factor to IS. In the surgical treatment group (n = 62) where Cobb angle > 40 the frequency of the PP genotype of ESR1 was significantly higher than in the controls (p = 0.023) and increased OR was observed (OR = 2.18; CI: ). The XX genotype was not significantly associated with curve severity (p > 0.05). The results are summarised in Table 2. T a b l e 1 RFLP protocol Gene, PCR Product Restriction Restriction Polymorphism Size, bp Enzyme Fragments, bp ESR1 rs XbaI ESR1 rs PvuII XX: 1374 Xx: xx: PP: 1374 Pp: pp: PCR indicates polymerase chain reaction; RFLP restriction fragment length polymorphism; bp base pair Compt. rend. Acad. bulg. Sci., 68, No 6,

4 T a b l e 2 Odds ratios of genotypes and alleles Subgroup Genotype, Allele p-value OR [95% CI] General group PP vs. Pp + pp [ ] P vs. p [ ] XX vs. Xx + xx [ ] X vs. x [ ] Cobb angle > 40 P vs. p [ ] PP vs. Pp + pp [ ] XX vs. Xx + xx [ ] X vs. x [ ] A value of p < 0.05 was considered to be statistically significant. OR indicates odds ratio; CI, confidence interval The analysis of all possible combinations of both polymorphisms revealed significant associations between two genotype combinations and genetic predisposition to IS and value of Cobb angle (Table 3). Discussion. Predisposition for IS, like other examples of complex traits, does not have a specific assigned risk of heritability, but inheritance is based on multiple factors, potentially both genetic and environmental [ 1 ]. In our study, the frequencies of the genotypes of PvuII polymorphism of ESR1 showed statistically significant differences between cases and controls and the PP genotype of ESR1 alone could be considered as a predisposing factor for IS. The P allele was associated with higher risk for development of IS. The genotype and allele frequencies of the XbaI polymorphism of ESR1 were comparable between cases and controls and the genotypes and alleles alone could not be considered as a susceptibility factor of IS. T a b l e 3 Odds ratios of genotype combinations XbaI-PvuII General group Genotype Combinations OR [95% CI] p-value XX-Pp 4.94 [ ] Xx-PP 4.38 [ ] Cobb angle > 40 XX-Pp 4.18 [ ] Xx-PP 3.23 [ ] A value of P < 0.05 was considered to be statistically significant. OR indicates odds ratio; CI, confidence interval 786 S. Nikolova, V. Yablanski, E. Vlaev et al.

5 Janusz et al. [ 6, 7 ] reported no statistically significant association between ESR1 common polymorphisms and occurrence or clinical form of IS in Polish females. The observed differences could be explained with the different allele frequencies in the different population and ethnic groups. The concept of disease-modifier genes as an element of genetic heterogeneity has been widely accepted and reported [ 1 ]. In the subgroup of surgical cases where Cobb angle > 40 a significant association between the PP genotype of ESR1 and IS was detected. The homozygous PP genotype of ESR1 could possess a modifying effect on the pathological phenotype. Inoue et al. concluded that the ESR1 XbaI polymorphism constitutes important factor for the curve progression [ 5 ]. Additionally, the frequency of the PvuII site polymorphism in patients with IS was not significantly different from controls [ 5 ]. The genotype combinations of XX-Pp and Xx-PP of ESR1 showed significantly elevated ORs. The hypothesis for synergistic effect of SNPs on the etiology and progression of IS has been widely accepted and reported [ 8 ]. In conclusion, ESR1 gene could be considered as susceptibility and modifying factor of IS. Our results suggest that the identification of molecular markers with diagnostic and prognostic value could facilitate the therapy decisions and early stage treatment of the patient with the least invasive procedures. An extended population-based case control study is necessary to confirm the contribution of these polymorphic variants to the development and progression of IS. REFERENCES [ 1 ] Cheng J. C., N. L. Tang, H. Y. Yeung, N. Miller (2007) Clin. Orthop. Relat. Res., 462, [ 2 ] Pensler J. M., J. A. Radosevich, R. Higbee, C. B. Langman (1990) J. Bone Miner. Res., 5, No 8, [ 3 ] Smith E. P., J. Boyd, G. R. Frank, H. Takahashi, R. M. Cohen, B. Specker, T. C. Williams, D. B. Lubahn, K. S. Korach (1994) N Engl. J. Med., 331, No 16, [ 4 ] Ivanova J., P. Doukova, M. Boyanov, P. Popivanov (2007) Hormones, 6, No 1, [ 5 ] Inoue M., S. Minami, Y. Nakata, H. Kitahara, Y. Otsuka, K. Isobe, M. Takaso, M. Tokunaga, S. Nishikawa, T. Maruta, H. Moriya (2002) Spine, 27, No 21, [ 6 ] Janusz P., T. Kotwicki, M. Andrusiewicz, M. Kotwicka (2013) PLoS One, 8, No 10, e Compt. rend. Acad. bulg. Sci., 68, No 6,

6 [ 7 ] Janusz P., M. Kotwicka, M. Andrusiewicz, D. Czaprowski, J. Czubak, T. Kotwicki (2014) BMC Musculoskelet. Disord., 15: 383. doi: / [ 8 ] Morocz M., A. Czibula, Z. B. Grozer, A. Szecsenyi, P. Z. Almos, I. Rasko, T. Illes (2011) Spine, 36, No 2, National Genetic Laboratory Medical University Sofia 2, Zdrave St 1431 Sofia, Bulgaria snikolova83@abv.bg University Orthopedic Hospital Prof. B. Boychev 56, Nikola Petkov Blvd 1614 Sofia, Bulgaria Orthopedic and Traumatology Clinic Tokuda Hospital 51B, Nikola Y. Vaptsarov Blvd 1407 Sofia, Bulgaria Molecular Medicine Center Medical University Sofia 2, Zdrave St 1431 Sofia, Bulgaria 788 S. Nikolova, V. Yablanski, E. Vlaev et al.

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