Short communication SUPEROXIDE DISMUTASE 2

CELLULAR & MOLECULAR BIOLOGY LETTERS http://www.cmbl.org.pl Received: 02 March 2015 Final form accepted: 20 August 2015 Published online:

Volume 21 (2016) pp 1-… DOI: 10.1515/cmble-2015-0041 © 2015 by the University of Wrocław, Poland

Short communication SUPEROXIDE DISMUTASE 2 POLYMORPHISMS AND OSTEOPOROSIS IN ASIAN INDIANS: A GENETIC ASSOCIATION ANALYSIS CHAITALI BOTRE1, ARJUN SHAHU1, NEERAJ ADKAR2, YOGESH SHOUCHE3, SAROJ GHASKADBI1 and RICHA ASHMA1* 1 Department of Zoology, Savitribai Phule Pune University, Pune – 411 007, Maharashtra, India, 2SaiShree Hospital and Joint Replacement Center, Aundh Pune – 411 007, India, 3Microbial Culture Collection, Indian Institute of Science Education and Research, Pune – 411008, India Abstract: Oxidative stress plays an important role in the development of osteoporosis. The present cross-sectional study focuses on mapping single nucleotide polymorphisms (SNPs) in the mitochondrial manganese superoxide dismutase (SOD2) gene in Asian Indians. The bone mineral density (BMD) of study subjects was assessed by dual x-ray absorptiometry. Individuals were classified as normal (n = 82) or osteoporotic (n = 98). Biochemical parameters such as vitamin D, total oxidant status (TOS) and SOD2 enzyme activity were estimated from plasma samples. Semi-quantitative PCR was carried out using GAPDH as an endogenous control. Genomic DNA was isolated from whole blood and SNPs were evaluated by PCR sequencing. Thirteen SNPs are reported in the examined region of the SOD2 gene, out of which in our samples SNPs rs5746094 and rs4880 were found to be polymorphic. Allele G of rs5746094 * Author for correspondence. Email: [email protected]; phone 91-02025601436 ext. 41; fax 91-20-25690617 Abbreviations used: BMD – bone mineral density; BMI – body mass index; cDNA – complementary deoxyribonucleic acid; CI – confidence interval; dbSNP – Single Nucleotide Polymorphism database; DNA – deoxyribonucleic acid; DXA – dual X-ray absorptiometry; ECLIA – electrochemiluminescence immunoassay; EDTA – ethylenediaminetetraacetic acid; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; GSR – glutathione S-reductase; HWE – Hardy-Weinberg equilibrium; ICMR – Indian Council of Medical Research; LD – linkage disequilibrium; MnSOD – manganese superoxide dismutase; MSA – multiple sequence alignment; OR – odds ratio; OS – oxidative stress; PBMCs – peripheral blood mononuclear cells; PCR – polymerase chain reaction; RNA – ribonucleic acid; ROS – reactive oxygen species; SNP – single nucleotide polymorphism; SOD2 – superoxide dismutase 2; TOS – total oxidant status; UTR – untranslated region

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(intronic) and allele C of rs4880 (exonic) are significantly higher in the osteoporotic individuals. Presence of allele C of rs4880 and increased level of TOS among osteoporotic individuals were found to be associated with disease risk. Keywords: Osteoporosis, Superoxide dismutase 2, MnSOD, rs4880, Oxidative stress, SNPs, Total oxidant status, rs5746094 INTRODUCTION The bone is a dynamic organ under continuous remodeling by osteoclasts that participate in bone resorption and osteoblasts that deposit new bone. This is a precisely balanced and coordinated process which is essential in the maintenance of bone homoeostasis. An imbalance in this process causes a difference in the bone turnover rate where the resorption surpasses the deposition. This leads to generalized skeletal fragility and an increase in the fracture risk incurred by the individual. It is a progressive condition called osteoporosis characterized by low bone mineral density (BMD). Osteoporosis has a complex etiology, and an individual’s risk of incurring a fracture is known to be associated with their genetic constitution and the environmental factors they are subjected to [1, 2]. The development of osteoporosis has been associated with decreasing levels of estrogen in postmenopausal women and the compensatory increase in follicle-stimulating hormone that leads to increased osteoclastogenesis [3]. Besides low estrogen level, decreased level of antioxidants also plays a pivotal role in the incidence of osteoporosis in aging individuals [4]. This leads to elevated levels of reactive oxygen species (ROS) being generated, particularly in osteoclasts, which then undergo differentiation and activation, leading to bone resorption [5, 6]. Oxidative stress (OS), an imbalance in the production of free radicals and the antioxidant defense of the cell, increases osteoclastogenesis [7]. Epidemiological studies have observed that there is an association between lower OS and higher BMD [8], and clinical studies have shown that dietary and/or supplemental ascorbate is associated with increased bone mass [9]. One of the key enzymes in the oxidative stress metabolism pathway is manganese superoxide dismutase (MnSOD) encoded by the SOD2 gene. MnSOD is a mitochondrial enzyme which converts superoxide to hydrogen peroxide. This is further removed by conversion to water and molecular oxygen by other antioxidant enzymes: catalase and glutathione peroxidase [10]. MnSOD is constitutively expressed in many cell types, including peripheral blood mononuclear cells (PBMCs) [11]. Mutations in the SOD2 gene have been linked to mitochondrial DNA damage leading to aging in fruit flies [12]. SOD2 knockout in mice is neonatal lethal [13] whereas heterozygous knockout mice exhibit 50% decreased SOD2 activity and increased oxidative stress and mitochondrial dysfunction [14]. In humans, polymorphisms in the SOD2 gene such as variant T of SNP rs4880 have been associated with cardiomyopathy in Japanese individuals [15], and other diseases, whereas variant C of the same


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SNP has been linked to hypertension [16] among other diseases. A study on European postmenopausal women showed no association of SNP rs4880 of the SOD2 gene to lumbar and femoral bone mineral density (BMD) [17]. Although polymorphisms in this gene have been linked to many diseases in Caucasians, their association with osteoporosis has not been explored in the Asian Indian population. The aim of this study is to assess the association of the SOD2 gene polymorphisms with osteoporosis in Asian Indian individuals. MATERIALS AND METHODS Sample collection The study was undertaken after being approved by the Institutional Ethics Committee. All procedures followed were in accordance with the ethics guidelines of the Indian Council of Medical Research (ICMR). A priori power analysis was carried out using G*Power 3.1.9.2 software, for one-tailed t-test with α-error set at 0.05 (statistical power of 95%), to detect medium effect size (ρ = 0.50). The total sample size (N) was computed to be 176 with the allocation ratio N2/N1. Bone density camps were arranged at different locations in Pune, Maharashtra to assess bone mineral density of individuals. All participants were informed about the study and written consent was obtained. Exclusion criteria followed while recruiting individuals for the study were the presence of any major diseases and oxidative stress-related diseases such as cardiovascular diseases, diabetes mellitus, hyper/hypotension, and bone-related diseases [20]. The questionnaire also included a section on physical activity and use of medications. Personal and family medical histories were collected with relevant clinical details such as age, sex, height, weight, body mass index (BMI), food habits (dietary intake of calcium) and lifestyle factors (smoking, alcohol intake) [20]. Smokers and alcoholic individuals were excluded from the study to evaluate the effect of oxidative stress purely due to osteoporosis in study groups. Peripheral blood (8-10 ml) was collected in vacutainers containing EDTA or sodium heparin sulfate as an anticoagulant (N = 180). Dual X-ray absorptiometry Bone density was measured by means of a portable dual X-ray absorptiometry (DXA). Peripheral-calcaneus BMD was assessed. This method has 90% sensitivity and specificity for osteoporosis as the gold standard measurement using DXA at the spine or proximal femur [18, 19]. Based on the BMD, unrelated individuals were classified as normal (n = 82) with T-score > -1.1 standard deviations, or osteoporotic (n = 98) with a T-score < -2.5 standard deviations. Biochemical estimations Plasma was extracted from blood containing EDTA or sodium heparin sulfate by centrifugation. Samples were stored at -80C until assays were performed. Vitamin D (25-OH-D) was assessed from plasma samples of normal and


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osteoporotic individuals by the electrochemiluminescence immunoassay (ECLIA) method using a COBAS e411 instrument from Roche Diagnostics, Germany at Genesys Laboratories, Pune, India. The circulating oxidant level was assessed by determining the total oxidant status (TOS) in plasma samples collected in heparin sulfate. In brief, TOS was estimated using Erel’s assay, which is a colorimetric method based on the oxidation of ferrous ion to ferric ion in the presence of various oxidative species in an acidic medium followed by measurement of the ferric ion with xylenol orange (Sigma Aldrich) [21]. The assay was calibrated by hydrogen peroxide (Merck Millipore, India) and the results were expressed as μmol H2O2/l. Estimation of superoxide dismutase enzyme levels in the plasma of normal and osteoporotic individuals was carried out by the tetrazolium salt method using the Superoxide Dismutase Activity Colorimetric Assay Kit (Cayman Chemicals, USA) as per the manufacturer’s instructions. RNA extraction and semi-quantitative PCR Peripheral blood mononuclear cells (PBMCs) were isolated from 8-10 ml of venous blood using Histopaque (Sigma Aldrich) by density gradient centrifugation and were frozen in Trizol (Invitrogen) within 3 hours of phlebotomy. Total RNA was isolated from the PBMCs of normal and osteoporotic individuals according to the method of Chomczynski and Sacchi (1987) [23]. Total RNA (1 μg) was converted to cDNA with a Verso cDNA Synthesis Kit (Thermo Scientific, USA). To obtain semiquantitative results, 1 μl of cDNA was PCR amplified in a total reaction volume of 25 μl using Taq Mastermix (NEB, USA) and 0.7 μM each of forward and reverse primers. Primers designed for the SOD2 gene were: forward (5′-TTTCAATAAGGAACGGGGACAC-3′) and reverse (5′-GTGCTCCCACACATCAATCC-3′). Initial denaturation was performed at 95°C for 5 min. Thirty-five cycles of amplification were performed at 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. A final extension at 72°C was carried out for 5 min. The primers for the endogenous control glyceraldehyde-3phosphate dehydrogenase (GAPDH) are as follows: forward (5′-TGGG GAAGGTGAAGGTCGGA-3′) and reverse (5′-GGGATCTCGCTGCTGGA AGA-3′). Initial denaturation was performed at 95°C for 5 min. Thirty-five cycles of amplification were performed at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. A final extension at 72°C was carried out for 5 min. All PCR products were analyzed by agarose gel electrophoresis, visualized by staining with ethidium bromide, and the gel bands were analyzed with ImageJ software (http://imagej.nih.gov/ij) [24]. DNA extraction and PCR sequencing Genomic DNA was extracted from venous blood by phenol-chloroform extraction and the ethanol precipitation method. PCR sequencing was carried out for a region of 563 bps including thirteen SNPs (Table 2). The primers were designed using the Primer3 (version 0.4.0) [22] online program for a specific



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region of the SOD2 gene. The sequences of primers were: forward (5′- ACTC GTGGCTGTGGTGGCTTCGGCA-3′) and reverse (5′-GGTACTTCTCCT CGGTGACGTTCA-3′). The amplicon obtained was 563 bp in length. The total reaction volume of 25 μl contained 25-30 ng of genomic DNA, 0.8 μM of forward and reverse primers each, 200 μM dNTP mix, 2 U of Taq polymerase and 1.5 mM MgCl2 (NEB, USA). Dimethyl sulfoxide was used as a PCR additive to a final concentration of 3%. The thermal cycling conditions were set as: initial denaturation at 95°C for 5 minutes, 40 cycles of denaturation 95°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s and final extension at 72°C for 5 minutes. PCR products were purified by polyethylene glycol and sodium chloride before being sequenced. DNA sequence reactions were done using BigDye Terminator v3.1 chemistry (Applied Biosystems, USA) and an ABI3700 capillary sequencer (Applied Biosystems, USA) according to the manufacturer's protocols. Software and statistical analysis The quality of chromatograms and trace score of sequences was checked using ABI SeqScanner (ver. 2.0) software. Multiple sequence alignment (MSA) was carried out using the ClustalW algorithm, which is part of the BioEdit (package ver. 7.0) [25]. Allele frequencies were calculated and Hardy-Weinberg equilibrium was studied. A chi-square test was performed to determine whether the allele frequencies of SNPs conformed to Hardy-Weinberg equilibrium (HWE). Linkage disequilibrium (LD) analysis was carried out between the polymorphic SNPs to determine the disequilibrium coefficient (D′) and correlation coefficient (r). The pair of SNPs was considered to be in high LD if D′ > 0.8 (D’ varies between 0 and 1) and r2 > 0.5 (varies between 0 and 1). Unpaired t- test was carried out to test association of polymorphic SNPs with disease status. A multinomial logistic regression test was performed for odds ratio (OR) calculation between the variables. The number of data points and predictor variables were set at 2 and 1 respectively. This non-linear regression test can have a p-value between 0 and 1. RESULTS AND DISCUSSION In the present study, normal and osteoporotic males and females of Asian Indian origin were recruited for polymorphism studies in the SOD2 gene region. A previous study by Mlakar et al. showed that point mutations in the intronic region of an antioxidant gene, the glutathione S-reductase gene (GSR; mutation GSR int3[A>G]) are associated with low BMD and SOD2 is differentially expressed in the circulating monocytes of premenopausal women with discordant bone mineral density (BMD) [17]. Thirteen SNPs were reported in the 5′ UTR (untranslated region) and partial coding sequence of the SOD2 gene. Both groups – normal (n = 82) and osteoporosis (n = 98) – had age-matched (45–75 years) males and females to verify the role of oxidative stress in the


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manifestation of osteoporosis. The overall male-to-female ratio was recorded separately for the study groups as 1:2.4 in the normal and 1:2 in the osteoporosis group. Anthropometric and biochemical parameters analysis BMI is known to be associated with BMD at different body sites [26]. In this study, the BMI between normal and osteoporotic individuals (N = 180) showed no significant difference (Table 1). Meta-analysis studies have shown that low weight and low BMI are related to an increased fracture risk. In individuals with a BMI of 20 kg/m2 there is a two-fold higher risk of fracture than individuals with higher BMI. Lower BMI in elderly women aged over 65 years has been correlated with an increased risk of hip fractures [26]. In the normal group, the number of vegetarians and non-vegetarians was equal, while in the osteoporosis group the vegetarians-to-non-vegetarians ratio was found to be 1:1.4. Oxidative stress is known to increase the levels of intracellular ROS, decreasing bone formation and inducing osteoclastogenesis [7, 27]. Oxidant molecules are produced endogenously through metabolic and physiological processes as well as due to interactions with the environment. Total peroxides and oxidative metabolites in serum and plasma samples will have additive effects and are therefore measured by total oxidant status (TOS) assay. The plasma samples obtained were assessed for TOS by Erel’s method [21]. It was found that total oxidant levels were significantly higher among osteoporotic individuals compared to normal ones (Table 1). This indicates that oxidants promote osteoclast differentiation and bone resorption over osteoblast formation in osteoporosis. We report here a significant difference in the TOS of age-matched normal and osteoporotic Asian Indians as also observed in other populations [28]. Furthermore, analysis for circulating vitamin D (25-hydroxy vitamin D) in the plasma samples of normal and osteoporosis groups showed a significant difference in the levels of vitamin D (Table 1). It was found to be largely insufficient in osteoporotic individuals, whereas it was borderline optimal among normal subjects. The Asian Indian population is noted to have lower levels of circulating vitamin D than Caucasian populations of Europe despite the adequate daylight hours and melanin pigmentation [29, 30]. Many have attributed this to inadequate exposure to sunlight and a deficient calcium diet, although studies in genetic epidemiology are currently ongoing [31]. SOD2 is transcriptionally and translationally regulated so the levels of the circulating enzyme and SOD2 mRNA are estimated [32, 33]. The plasma samples obtained from the blood of normal and osteoporotic individuals were also evaluated for SOD2 enzyme levels. However, it was observed that the SOD2 enzyme was not significantly different between the two groups (Table 1). To further confirm this, semi-quantitative analysis was carried out.



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Table 1. Comparison of anthropometric and biochemical parameters of normal and osteoporotic individuals in the age group 45-75 years. Data are represented as mean ± SD. Age (years)

Normal 57.4 ± 8.51

Osteoporosis 60.20 ± 7.42

T-value 1.5303

p* 0.1294

BMI (kg/m2)

26.6 ± 4.4

25.4 ± 4.0

1.0914

0.2786

TOS (μmol H2O2/l)

37.23 ± 13.8

53.03 ± 16.87

5.3578

0.0001

25-OH-D (mg/dl)

23.45 ± 9.34

14.38 ± 7.27

3.8022

0.0004

SOD2 (U/ml)

0.063 ± 0.02

0.0763 ± 0.0432

1.8199

0.0731

Semi-quantitative PCR analysis of SOD2 mRNA level Semiquantitative PCR for the SOD2 gene was carried out in triplicate using GAPDH as an endogenous control. Our results indicate no significant difference between the transcript levels of SOD2 in normal and osteoporosis samples obtained from PBMCs (Fig. 1). Despite the high TOS in osteoporotic individuals, SOD2 enzyme and transcript levels show no significant difference. Further SNPs were investigated in the SOD2 gene by sequence analysis between the two groups as variations in the coding region may not necessarily affect the protein’s activity but may affect its subcellular localization [34].

Fig. 1. Semi-quantitative PCR of MnSOD gene from PBMCs of osteoporosis and normal individuals.

Allele frequency distribution in the 5′ UTR and coding region of the SOD2 gene The Asian Indian population has not previously been studied for SNP variation in the SOD2 gene. PCR sequencing of the 5′ UTR and coding region of SOD2 was carried out to include exon 1, intron 1 and exon 2 regions. In this region, 13 SNPs have been reported in the dbSNP and HapMap databases (Table 2). In Asian Indians (from Pune, Maharashtra), direct sequencing did not reveal any previously unknown SNPs. In the sampled population, sequence and


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chromatogram analysis of the DNA from peripheral leukocytes showed eleven monomorphic SNPs and two polymorphic SNPs (rs5746094 and rs4880) which were further analyzed (Fig. 2). SNPs rs5746094 and rs4880 are separated by 296 bp and linkage disequilibrium for the two polymorphic loci is 0.08 (Lewontin’s D′, D′ > 0; r2 = 0.357), suggesting that the allele of one locus does not influence the allele distribution pattern of the other locus. Hardy-Weinberg equilibrium (HWE) was calculated and the χ2 test was performed with 1 degree of freedom. The study groups conformed to HWE (Table 3). Table 2. Details of selected SNPs within SOD2 5′UTR and coding region (from dbSNP). SNP reference sequence No.

Region

rs374949337

5′UTR

Positions Global minor HeteroAncestral w.r.t. ATG allele frequency/ zygosity allele (+1) minor allele count -58 NA NA C

rs199543686 rs79988672

Intron 1

Reference (dbSNP) NA

-47

NA

NA

C

NA

+23

NA

NA

G

NA

rs5746094

+31

0.408

C = 0.2736 / 1370

A

1000Genomes

rs5746095

+42

0.004

G = 0.0010/5

G

1000Genomes

rs2758342

+49

NA

NA

C

NA

rs2758341

+50

NA

NA

G

NA

rs201297522

+280

NA

C = 0.0012/6

G

1000Genomes

rs77799497 rs5746096

Exon 2

+302

NA

NA

A

NA

+309

0.005

A = 0.0014/7

G

1000Genomes

rs372258485

+324

NA

NA

G

NA

rs4880

+327

0.466

G = 0.4107/2057

C

1000Genomes

rs375506679

+363

NA

NA

NA

NA

NA: not available

Table 3. Genotype and allele frequencies at the polymorphic SNPs of the SOD2 gene in the investigated population with chi-square tests of goodness-of-fit for Hardy-Weinberg equilibrium law and 2X2 contingency chi-square for genetic association analysis. SNP

Condition

Genotype frequencies CC

rs4880

rs5746094

TT

C

T

Normal (n = 82)

0.2601

0.4998

0.2401

0.51

0.49

0.9989

Osteoporosis (n = 98)

0.3721

0.4758

0.1521

0.61a

0.39

0.9577

AA

AG

GG

A

G

Normal (n = 82)

0.4096

0.4608

0.1296

0.64

0.36

0.9406

0.29

b

0.8877

Osteoporosis (n = 98) a

CT

P-value (χ2, HWE)

Allele frequencies

0.0841

0.4118

0.5041

0.71

2X2 contingency test: χ2 = 19.908, P < 0.0001, b 2X2 contingency test: χ2 = 24.6206, P < 0.001



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Fig. 2. Sequence analysis of SOD2 gene region showing polymorphic SNPs – rs5746094 (A–C) and rs4880 (D–F).

The genotype and allele frequency distribution across these two SNPs (Table 3) indicates that allele G of rs5746094 is significantly higher in osteoporotic individuals than in normal ones. McNemar’s 2x2 contingency test using a multiplicative genetic model based on alleles was used for odds ratio estimation [36], which showed SOD2-associated osteoporosis risk for the G allele [OR = 0.23; 95% confidence interval (CI) 1.15-3.09]. This intronic SNP has not previously been associated with other diseases or alterations in gene expression and protein function. Whether it influences osteoporosis occurrence needs to be further investigated. Similarly, allele C of rs4880 is significantly higher in the osteoporosis group, with a high OR [OR = 1.50; 95% CI 0.85-2.63]. Thus, presence of variant C with higher frequency among osteoporotic individuals with high OR indicates disease risk conferred by allele C of rs4880. To further understand the etiology of osteoporosis, a multinomial logistic regression test for allele C of rs4880 and TOS among disease and normal subjects was performed based on computed values (Tables 1 and 3). The odds ratio obtained (Table 4) suggests a possible synergistic role of TOS and allele C of rs4880 in the occurrence of osteoporosis. However, this needs to be further confirmed.


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Table 4. Multinomial logistic regression test for allele C of rs4880 and TOS among disease and normal subjects. TOS (μmol H2O2/l)

Normal 37.23 ± 13.8

Osteoporosis 53.03 ± 16.87

C-allele frequency

51.2

61.2

Odds ratio

95% confidence interval

TOS (μmol H2O2/l)

1.19

Lower limit 0.68

Upper limit 2.09

C-allele frequency

0.83

0.47

1.46

SNP variation analysis for disease susceptibility in osteoporosis Rs4880 is present in the second exon of SOD2 and is known to play an important role in SOD2 protein structure alteration and function and therefore in disease occurrence. It has been previously reported to be associated with cardiomyopathy [15], hypertension [16], prostate and other cancers [34]. The transition variation of this SNP affects the 16th amino acid of MnSOD which replaces alanine (GCT) with a valine (GTT) (A16V) in the coding sequence, causing a nonsynonymous mutation. Differential subcellular localization of SOD2 due to rs4880 had been experimentally demonstrated by ShimodaMatsubayashi and colleagues (1996) [37]. This variation is present in the mitochondrial targeting sequence, and the presence of valine impairs the import of translated mRNA into the mitochondrial matrix [35]. CONCLUSION Thus, the present cross-sectional study examines SNPs of the SOD2 gene among normal and osteoporotic individuals. Out of the total 13 reported SNPs in the selected region of the gene, only two SNPs (rs5746094 and rs4880) were found to be polymorphic. Association analyses were performed on variant C of rs4880. This allele was found to be significantly higher in the osteoporotic individuals along with TOS. Odds ratio estimation suggests association of TOS and allele C with osteoporosis. Our findings support the hypothesis that oxidative stress contributes to occurrence of osteoporosis, a complex life style disease. Although this study is cross-sectional and the samples are representative of the Asian Indian population, a larger sample size is required to increase the statistical power of the present association analysis. Acknowledgments. The authors thank the Department of Zoology, Savitribai Phule Pune University, for providing infrastructure and equipment facilities and the funding agencies - the Department of Biotechnology (DBT), University Grants Commission – Center for Advanced Studies (UGC – CAS) grant, University with Potential of Excellence phase II grant, Department of Science and Technology – Promotion of University Research and Scientific Excellence



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(DST – PURSE) for financial support. We are grateful to the volunteers and osteoporosis patients for their participation in this ongoing study. Conflict of interest. The authors declare no potential conflict of interest related to this article. Author contributions. R.A and S.G designed the study. C.B. and A.S. performed the experiments. C.B. and R.A. wrote the manuscript. Y.S and S.G. reviewed and edited the manuscript. N.A. helped in arranging bone mineral density test camps in and around the Pune district of Maharashtra. Y.S. also helped in sequencing of samples. REFERENCES 1. Ralston, S.H. and de Crombrugghe, B. Genetic regulation of bone mass and susceptibility to osteoporosis. Genes Dev. 20 (2006) 2492-2506. 2. Ferrari, S.L. Osteoporosis: A complex disorder of aging with multiple genetic and environmental determinants. World Rev. Nutr. Diet. 95 (2006) 35-51. 3. Sun, L., Peng, Y., Sharrow, A.C., Iqbal, J., Zhang, Z., Papachristou, D.J. and Zaidi, M. FSH directly regulates bone mass. Cell. 125 (2006) 247-260. 4. Lean, J.M., Davies, J.T., Fuller, K., Jagger, C.J., Kirstein, B., Partington, G.A., and Chambers, T.J. A crucial role for thiol antioxidants in estrogendeficiency bone loss. J. Clin. Invest. 112 (2006) 915-923. 5. Baek, K.H., Oh, K.W., Lee, W.Y., Lee, S.S., Kim, M.K., Kwon, H.S. and Kang, M.I. Association of oxidative stress with postmenopausal osteoporosis and the effects of hydrogen peroxide on osteoclast formation in human bone marrow cell cultures. Calcif. Tissue Int. 87 (2010) 226-235. 6. Srinivasan, S., Koenigstein, A., Joseph, J., Sun, L., Kalyanaraman, B., Zaidi, M. and Avadhani, N.G. Role of mitochondrial reactive oxygen species in osteoclast differentiation. Ann. N. Y. Acad. Sci. 1192 (2010) 245-252. 7. Manolagas, S.C. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr. Rev. 31 (2010) 266-300. 8. Maggio, D., Barabani, M., Pierandrei, M., Polidori, M.C., Catani, M., Mecocci, P. and Cherubini, A. Marked decrease in plasma antioxidants in aged osteoporotic women: results of a cross-sectional study. J. Clin. Endocr. Metab. 88 (2003) 1523-1527. 9. Morton, D.J., Barrett‐Connor, E.L. and Schneider, D.L. Vitamin C supplement use and bone mineral density in postmenopausal women. J. Bone Miner. Res. 16 (2001) 135-140. 10. Mao, G.D., Thomas, P.D., Lopaschuk, G.D. and Poznansky, M.J. Superoxide dismutase SOD-catalase conjugates - Role of hydrogen peroxide and the Fenton reaction in SOD toxicity. J. Biol. Chem. 268 (1993) 416-420. 11. Dernbach, E., Urbich, C., Brandes, R.P., Hofmann, W.K., Zeiher, A.M. and Dimmeler, S. Antioxidative stress–associated genes in circulating progenitor


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12. 13.

14.

15.

16. 17.

18. 19.

20. 21. 22. 23. 24.

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cells: evidence for enhanced resistance against oxidative stress. Blood 104 (2004) 3591-3597. Sun, J., Folk, D., Bradley, T.J. and Tower J. Induced overexpression of mitochondrial Mn-superoxide dismutase extends the life span of adult Drosophila melanogaster. Genetics 161 (2002) 661-672. Lebovitz, R.M., Zhang, H., Vogel, H., Cartwright, J., Dionne, L., Lu, N. and Matzuk, M.M. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc. Natl. Acad. Sci. 93 (1996) 9782-9787. Williams, M.D., Van Remmen, H., Conrad, C.C., Huang, T.T., Epstein, C.J. and Richardson, A. Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J. Biol. Chem. 273 (1998) 28510-28515. Hiroi, S., Harada, H., Nishi, H., Satoh, M., Nagai, R. and Kimura, A. Polymorphisms in the SOD2 and HLA-DRB1 Genes are associated with nonfamilial idiopathic dilated cardiomyopathy in Japanese. Biochem. Biophys. Res. Commun. 261 (1999) 332-339. Hsueh, Y.M., Lin, P., Chen, H.W., Shiue, H.S., Chung, C.J., Tsai, C.T. and Chen, C.J. Genetic polymorphisms of oxidative and antioxidant enzymes and arsenic-related hypertension. J. Tox. Env. Health [A] 68 (2005) 1471-1484. Mlakar, S.J., Osredkar, J., Prezelj, J. and Marc, J. Antioxidant enzymes GSR, SOD1, SOD2, and CAT gene variants and bone mineral density values in postmenopausal women: a genetic association analysis. Menopause 19 (2012) 368-376. McCauley, E., Mackie, A., Elliott, D. and Chuck, A. Heel bone densitometry: device specific thresholds for the assessment of osteoporosis. The Brit. J. Radiol. 79 (2006) 464-467. Sweeney, A.T., Malabanan, A.O., Blake, M.A., Weinberg, J., Turner, A., Ray, P. and Holick, M.F. Bone mineral density assessment: comparison of dual-energy X-ray absorptiometry measurements at the calcaneus, spine, and hip. J. Clin. Densitom. 5 (2002) 57-62. McClung, M.R. Clinical risk factors and evaluation of the risk of osteoporosis in clinical practice. Ann. Med. Interne. Paris 151 (2000) 392-398. Erel, O. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem. 37 (2004) 112-119. Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159. Schneider, C.A., Rasband, W.S., Eliceiri, K.W., Schindelin, J., ArgandaCarreras, I., Frise, E. and Roysam, B. NIH image to ImageJ: 25 years of image analysis. Nat. Methods. 9 (2012) 671-675. Rozen, S. and Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132 (2000) 356-386.



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25. Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. (Ser.Oxf.) 41 (1999) 95-98. 26. Morin, S., Tsang, J.F. and Leslie, W.D. Weight and body mass index predict bone mineral density and fractures in women aged 40 to 59 years, Osteoporos. Int. 20 (2009) 363-370. 27. Salahuddin, P., Rabbani, G. and Khan, R. The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach. Cell. Mol. Biol. Lett. 19 (2014) 407-437. 28. Altindag, O., Erel, O., Soran, N., Celik, H. and Selek, S. Total oxidative/ anti-oxidative status and relation to bone mineral density in osteoporosis. Rheumatol. Int. 28 (2008) 317-321. 29. Sánchez-Rodríguez, M.A., Ruiz-Ramos, M., Correa-Muñoz, E. and Mendoza-Núñez, V.M. Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskelet. Disord. 8 (2007) 124. 30. Arya, V., Bhambri, R., Godbole, M.M. and Mithal, A. Vitamin D status and its relationship with bone mineral density in healthy Asian Indians. Osteoporos. Int. 15 (2004) 56-61. 31. Awumey, E.M., Mitra, D.A., Hollis, B.W., Kumar, R. and Bell, N.H. Vitamin D metabolism is altered in Asian Indians in the southern United States: A clinical research center study. J. Clin. Endocr. Metab. 83 (1998) 169-173. 32. Mithal, A., Wahl, D.A., Bonjour, J.P., Burckhardt, P., Dawson-Hughes, B., Eisman, J.A. and Morales-Torres, J. Global vitamin D status and determinants of hypovitaminosis D. Osteoporos. Int. 20 (2009) 1807-1820. 33. Tao, R., Coleman, M.C., Pennington, J.D., Ozden, O., Park, S.H., Jiang, H. and Gius, D. Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress. Mol. Cell. 40 (2010) 893-904. 34. Crawford, A., Fassett, R.G., Geraghty, D.P., Kunde, D.A., Ball, M.J., Robertson, I.K., and Coombes, J.S. Relationships between single nucleotide polymorphisms of antioxidant enzymes and disease. Gene 501 (2012) 89–103. 35. Sutton, A., Imbert, A., Igoudjil, A., Descatoire, V., Cazanave, S., Pessayre, D. and Degoul, F. The manganese superoxide dismutase Ala16Val dimorphism modulates both mitochondrial import and mRNA stability. Pharmacogenet. Genomics 15 (2005) 311-319. 36. Lewis, C.M. Genetic association studies: design, analysis and interpretation. Brief. Bioinform. 3 (2002) 146-153. 37. Shimoda-Matsubayashi, S., Matsumine, H., Kobayashi, T., NakagawaHattori, Y., Shimizu, Y. and Mizuno, Y. Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene: a predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson's disease. Biochem. Biophys. Res. Commun. 226 (1996) 561-565.
