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Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population
GENREP-00032; No of Pages 5 Gene Reports xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Gene Reports journal homepage: www.elsevier.com/locate/genrep
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Krishna Subramanyam a,1, Subhadra Poornima b,1, Keerthi Konda Juturu b, Dev Anand c, Shruthy Mohanthy d, Imran Ali khan e, Qurratulain Hasan b,⁎
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Article history: Received 26 February 2016 Received in revised form 30 April 2016 Accepted 3 May 2016 Available online xxxx
Department of Orthopedics, Yashoda Hospitals, Hyderabad 500036, India Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad 500068, India Department of Orthopedics, Kamineni Institute of Medical Sciences, Nalgonda 508254, India d Department of Biochemistry, Kamineni Institute of Medical Sciences, Nalgonda 508254, India e Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, P.O. Box-10219, King Saud University, Riyadh 11433, Saudi Arabia
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Osteoarthritis (OA) is a common and multifactorial skeletal disabling disease in the adult/elder population. Vitamin D was involved with multiple diseases including OA. Based on the prior studies, the single nucleotide polymorphism in the vitamin D receptor (VDR) gene might be a risk factor for the development of autoimmune diseases. However, we aimed to investigate whether FokI polymorphism and serum vitamin D levels are in association with primary knee OA in south Indian population. In this case–control study we have selected 100 OA cases and 100 healthy controls from Kamineni Hospitals in Telangana and India. The collected blood samples were used for estimation of serum vitamin D levels and isolation of genomic DNA. PCR was carried out with specific primers. The FokI enzyme was used to perform RFLP and followed by electrophoresis and analysis. The clinical data has been calculated separately between the OA cases and controls. The biochemical analysis performed with ELISA method revealed that 44% of cases were vitamin D deficient, 28% had in sufficient vitamin D levels and 28% had sufficient vitamin D levels. We have performed the statistical analysis between the OA cases and controls and we could not find any significant association either with allele and genotype frequencies (p N 0.05). The gender association analysis also failed to disclose the significant difference between males and females (p N 0.05). The results of our present study concludethat genetic missense variant of FokI in VDR gene does not associate with primary knee OA in the south Indian population. © 2016 Published by Elsevier Inc.
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Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population
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Keywords: Osteoarthritis VDR gene FokI polymorphism Asian Indian population
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1. Introduction
Osteoarthritis (OA) is a common chronic and multifactorial etiology with a group of mechanical abnormalities involving degradation of joints and subchondral bone (He et al., 2014). The prevalence of OA varies widely between studies and depends on the study population and increases with age with osteoarthritic changes uncommon under the age of 40, but seen in most people over the age of 70. Obesity and overweight are the major factors that became rapidly growing threat to worldwide health and the co morbidities include hypertension, type2 diabetes, stroke and cardiovascular diseases.
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Abbreviations: OA, Osteoarthritis; VDR, Vitamin D Receptor; PCR, Polymerase chain reaction; RFLP, Restriction Fragment Length Polymorphism; ELISA, Enzyme Linked Immunosorbent Assay; SNP, Single Nucleotide Polymorphism; SNPs, Single Nucleotide Polymorphisms; K/L Grade, Kellgren/Lawrence Grade; 25OHVD, 25-hydroxyvitamin D; OR, Odds Ratio; CIs, Class Intervals. ⁎ Corresponding author at: Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad 500068, Telangana, India. E-mail address: [email protected] (Q. Hasan). 1 Equal contribution.
The knee, hip and hand are most affected by the disease (Van Schoor et al., 2015). Knee OA is the commonest degenerative arthritis. Knee OA is a mechanically driven disease and it is suggested that medial tibiofemoral knee-joint load increases with pharmacologic pain relief, indicating that it may be positively associated with disease progression. The underlying mechanisms are still unknown; a genetic component has been established by population studies. Genetic factors contribute to the development of OA the heritability has been estimated at 48%– 65% (Ai et al., 2014). The World Health Organization Scientific Group on Rheumatic Diseases estimates that among those people who are 60 or older, the incidence rate of OA is up to 1 in 10 (Pereira et al., 2011). Age, sex, obesity, ethnicity and profession are the multiple risk factors and well recognized environmental factors, genetic variations have also been found to be strong determinants (Cai et al., 2014). The involvement of vitamin D in bone and cartilage metabolisms could explain why alterations in vitamin D homeostasis are associated to several pathological conditions of knee cartilage and intervertebral disc tissue, in particular OA and disc degeneration linked pathologies (Colombini et al., 2013). Single nucleotide polymorphisms (SNPs) are single base variations between genomes, useful for genome-wide
http://dx.doi.org/10.1016/j.genrep.2016.05.001 2452-0144/© 2016 Published by Elsevier Inc.
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Medicine, Kamineni hospitals (Khan et al., 2015). The genomic DNA was quantified with NanoDrop and stored at −80 °C until further use. Genotyping determination for fok1 polymorphism in the VDR gene, located in exon 2 was carried out with polymerase chain reaction and restriction fragment length polymorphism performed with 2% agarose gel electrophoresis.
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2.5. Statistical Analysis
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2. Materials and methods
The association of genotype in OA was examined by odds ratio analysis with 95% confidence interval using Openepi software (Khan et al., 2014). The allele frequencies of VDR gene were tested to determine whether they were in accordance with HWE (Hardy–Weinberg equilibrium). Clinical data are expressed as mean ± standard deviation. Data analysed by ANOVA. An independent t-test was used to test the case and controls. Statistical significance was defined as p b 0.05.
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2.1. Ethics
3. Results
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3.1. Demographic details of the patients and controls
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Ethical approval for this current research was obtained from the Institutional Ethics Committee, Kamineni Hospitals, Hyderabad, India. Data and blood collection from study individuals were collected after taking their informed consent.
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2.2. Patient recruitment
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The hospital based case–control study has been carried out in one hundred (n = 100) south Indian OA patients who were clinically diagnosed and radiologically confirmed with primary knee OA and 100 healthy controls were arbitrarily selected from the routine master health checkup examination who did not have any complaints of OA or autoimmune diseases. The selection, inclusion and exclusion criteria of cases and controls have been published in our earlier paper (Poornima et al., 2015). All the patients were recruited from Department of Orthopedics, Kamineni hospitals, Hyderabad, India. The age range of the patients was 25–60 years (42.4 ± 8.1) and the age range of controls was 26–62 (42.1 ± 7.9). Personal, medical and family histories were documented in a well-designed proforma.
The clinical features of cases and controls have been described in Table 1. The mean age of the cases was 42.4 ± 8.1 and controls was 42.1 ± 7.9, which are similar (p = 0.87). The clinical features such as age, gender and height were also similar between cases and controls. The weight and BMI of cases was significantly greater (p b 0.05). Comorbid factors hypertension, thyroid dysfunction and T2DM were more in the cases compared to controls (p b 0.05). In this study, 28% of the cases had positive family history of OA.
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2.3. OA score
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The radiographic image and clinical information were used for the Kellgren/Lawrence (K/L) score (0–4 scale). K/L system is a method of classifying the severity of knee osteoarthritis using five grades (Kellgren and Lawrence, 1957)
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1. Grade 0: No radiographic features of Osteoarthritis
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2. Grade 1: Doubtful joint space narrowing and possible osteophytic lipping. 3. Grade 2: Definite osteophytes and possible joint space narrowing on anteroposterior weight bearing radiograph 4. Grade 3: Multiple osteophytes, definite joint space narrowing, sclerosispossible bony deformity. 6. Grade 4: Large osteophytes marked joint space narrowing, severe sclerosis and definitely bony deformity.
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2.4. Investigation of FokI polymorphism
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Four milliliters of the peripheral blood was collected and 2 mL of the serum was used for measurement of 25-hydroxyvitamin D (25 OH VD) levels, by ELISA method using IDA kit manufactured by Immunodiagnostic Systems Ltd., UK. Individuals were categorized as deficient (b20 ng/ml), insufficient (20–30 ng/ml), sufficient (30–100 ng/ml) and potential toxic (N100 ng/ml). The remaining 2 mL of the blood was used for genomic DNA extraction using salting out technique, the standard method used in the Department of Genetics and Molecular
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3.2. Biochemical analysis
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mapping and study of disease genes. SNP is a useful marker for diseases in haplotype-based association studies and in linkage disequilibrium analysis (Khan et al., 2016). Ng and Henikoff (2001) have predicted the effect of SNPs on protein function based on sequence homology methods and many polymorphisms in vitamin D Receptor (VDR) were identified. VDR gene covers 105 kb, with an extensive promoter region capable of generating multiple tissue-specific transcripts (Colombini et al., 2013). VDR harbors several known functional polymorphisms and some of these have been investigated (He and Wang, 2015). Several studies investigated the role of VDR (FokI)/rs2228570 polymorphism in the susceptibility to common diseases such as T2DM, osteoporosis and OA. Based on the prior studies, we have carried out our study to investigate whether FokI polymorphism and serum vitamin D levels are in association with south Indian population.
The serum concentration of 25 OH VD was measured using ELISA method. This test was performed for the identification of regulation of bone metabolism and calcium homeostasis effects as a deficiency or excess of vitamin D. We have measured the vitamin D levels in knee osteoarthritic cases. The results of our study showed that 44% of cases were vitamin D deficient, 28% had in sufficient vitamin D levels and 28% had sufficient vitamin D levels.
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3.3. Polymorphism analysis
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The genotype and allele distribution of Fok1 variants in OA cases and controls is shown in Table 2. Genotype distribution deviated from the HWE with total absence of TT genotype in both cases and controls. The genotype distribution in OA cases was 95% in AA and 5% in AT, whereas in controls, the genotypic distribution was 99% in AA and 1% in AT. The allele frequencies between the cases and controls did not showed significant difference i.e. T vs A: OR-5.1 (95% CI: 0.5–44.0); p = 0.13. Both dominant and recessive mode of inheritance did not show significant difference between cases and controls. Statistically we could not find any significance among our cases and controls.
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Table 1 Demographic details of the patients and controls.
t1:1 t1:2
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Characters
Cases (n = 100)
Controls (n = 100)
p value
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Age (years) Sex: (M:F) Height (cm) Weight (kg) BMI (kg/m2) Age of onset Family history of OA History of HTN History of T2DM History of thyroid dysfunction
42.41 ± 8.11 32:68 156.63 ± 2.68 76.97 ± 8.44 31.38 ± 3.4 39.52 ± 6.89 28 (28%) 50 (50%) 38 (38%) 39 (39%)
42.17 ± 7.98 31:69 155.46 ± 2.45 69.78 ± 7.87 25.89 ± 2.67 NA NA 15 (15%) 14 (14%) 22 (22%)
0.87 0.88 0.37 0.001 0.01 NA NA b0.01 b0.01 b0.01
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
K. Subramanyam et al. / Gene Reports xxx (2016) xxx–xxx Table 2 Genotype and allele distribution of VDR-Fok1 gene polymorphism.
t2:3
Genotype
OA cases
Controls
OR 95% CI
t2:4 t2:5 t2:6 t2:7 t2:8
CC CT TT C T
95 (95%) 05(5%) 0 (0%) 195 (0.975) 05 (0.025)
99 (99%) 01 (1%) 0(0%) 199 (0.995) 01 (0.005)
1.00 5.2 (0.5–45.40) 3.1 (0.61–15.87) 1.00 5.1 (0.5–44.5)
p value
Vitamin D levels
CC genotype
CT genotype
TT genotype
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0.13 0.16
Deficient Insufficient Sufficient Toxic
43% 34% 23% 0
60% 20% 20% 0
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0.13
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3.4. Statistical correlation between genders
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We tried to correlate the OA cases with the gender subjects, i.e. males (32%) vs. females (68%). The genotype and allele frequency of A25920T variants with gender have been documented in Table 3. Genotypic and allelic distribution did not show any statistical significance between males and females i.e. T vs A: OR-2.1 (95% CI: 0.12–4.78); p = 0.56. (See Table 4.)
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3.5. Correlation between vitamin D levels and VDR genotype in cases
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In this study we found deficiency, insufficiency and sufficiency of vitamin D levels. 43% of the cases were deficient vitamin D levels with AA genotype, 34% had insufficient vitamin D with AA genotype and 23% had sufficient vitamin D levels with homozygous AA genotype. We found 60% cases with AT genotype in which 3 cases had deficient vitamin D, and two cases had sufficient vitamin D levels (Fig 1). When we compared the vitamin D levels of OA cases and degenerative spine arthritis cases with random controls of general population the percentage of individuals who had deficient vitamin D levels was higher (Table 5).
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4. Discussion
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The aim of this study was to identify the association between FokI polymorphism of VDR gene in primary knee OA in Asian Indian population. Our results were displayed as negative association and we could not find the statistical significance between cases and controls. Serum 25-hydroxyvitamin D (25OHD) concentration is an effective indicator of vitamin D status in humans (Jia et al., 2015). Most of the cases exposed the deficiency of vitamin D levels (44%) and 28% of cases were insufficiency and 28% had sufficient vitamin D levels. Deficient levels of vitamin D levels in an individual with a variant allele are associated with the diseases. One of our unpublished data in our cohort showed statistical significance between spine degenerative cases and healthy controls. VDR gene polymorphism may be associated with the smaller joints like spine but not like knee joints. In our study, we found 5 heterozygous CT VDR genotype cases, 4 were obese and 1 were overweight with respect to BMI, which is indicating obesity with heterozygous genotype individuals are more prone to the disease. An evidence that osteoarthritis, diabetes, cancer, cardiovascular diseases, tuberculosis,
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Table 3 Genotype and allele frequencies of VDR-Fok1 gene polymorphism between males and females.
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virus infections, urinary stones, periodontitis, and autoimmune diseases have been reported with FokI polymorphism in VDR gene might be associated with the development of autoimmune diseases (Zilahi et al., 2015). VDR polymorphism, such as ApaI (rs7975232), Taq1 (rs731236), Bsm1 (rs1544410) and FokI (rs2228570) have been reported to be associated with various diseases. FokI located in exon 2, results in different translation initiation sites on VDR gene. This is the only known VDR polymorphism that translates in different protein products (Uitterlinden et al., 2004). Earlier studies reported the association between VDR polymorphisms and obesity (Vasilopoulos et al., 2013) for different ethnic group (Binh et al., 2011) and for type 2 diabetic subjects (Ye et al., 2001). Earlier studies have showed the positive (Vasilopoulos et al., 2013) and negative (Huang et al., 2000; Colombini et al., 2013; Valdes et al., 2004; Uitterlinden et al., 1997) association with VDR gene and OA. FokI genetic variant was positively associated with Broncho pulmonary dysplasia, T2DM, pre/post-menopausal women and falls, balance and muscle power (Koroglu et al., 2014; Al-Daghri et al., 2012; Yasovanthi et al., 2011; Barr et al., 2010). Liu et al. (2014) from their meta-analysis studies concluded that the variants in the VDR gene such as Bsm1, Taq1 and Apa1 might not be an important predictor for OA. Zhu et al. (2014) studies also performed the meta-analysis studies was designed between the Europe versus Asian subjects and their results suggested that VDR gene polymorphism play a role in OA aetiology in the Asian population. Our results were not in accordance with the above supported articles. Bid et al. (2005) had carried out the VDR gene polymorphisms such as FokI, TaqI and ApaI in normal healthy controls and this data has compared with other ethnicity results and concluded that the frequency and distribution of the polymorphisms in India are substantially different from other ethnic population groups. The functional effects of the VDR TaqI polymorphism is located near the 3′-untranslated region i.e. in intron 8/exon 9. Human VDR gene is mapped at chromosome 12q12-q14, and five common polymorphisms have been typically associated with VDR activity, namely VDR Tru9I (rs757343), FokI (rs2228570), TaqI (rs731236), BsmI (rs1544410) and ApaI (rs7975232). Few studies have suggested that these polymorphisms might alter polyadenylation of the VDR mRNA transcript, and thus affect mRNA stability. VDR protein and gene plays an important role in vitamin D pathway and it is an intracellular hormone receptor that specifically binds the biologically active form of calcitriol or vitamin D, 1,25-dihydroxyvitamin D and interacts with specific nucleotide sequences of target genes to produce a variety of biologic effects and involves conversion of serum 25-hydroxyvitamin D into active hormone, 1,25-dihydroxyvitamin D. The protein in VDR is known to display polymorphic variation, belongs to the steroid hormone family of nuclear receptors which are responsible for the transcriptional regulation of a number of hormone responsive genes. (Uitterlinden et al., 2004; He and Wang, 2015).
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However one of our unpublished data revealed that recessive mode of inheritance showed significant association between degenerative spine arthritic cases and controls (4.25 95% CI {1.62–11.14} p = 0.003).
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Table 4 Correlation between vitamin D levels and VDR genotype in OA cases.
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Genotype OA males (n = 32) OA females (n = 68) OR 95% CI
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CT TT C T
01(3%) 00 (0%) 63 (0.99) 01 (0.01)
04 (6%) 00 (0%) 132(0.97) 04 (0.03)
2.12 (0.12–35.06) 0.50 (0.05–4.73) 0.83 (0.15–4.49) Reference 2.12 (0.12–4.78)
0.55 0.82 0.56
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Table 5 t5:1 Comparison of vitamin D levels between spine degenerative arthritis cases, osteoarthritis t5:2 cases and random healthy controls. t5:3 Vitamin D levels
Degenerative spine arthritic cases
OA cases
Random healthy controls
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Deficiency Insufficiency Sufficiency Toxic
43% 34% 23% 0
44% 28% 28% 0
52% 29% 19% 0
t5:6 t5:7 t5:8 t5:9
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Conflict of interest
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All the authors declare that there is no conflict of interests towards this article.
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Acknowledgement
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We are thankful to the patients for their support towards this study and we are thankful to Kamineni hospitals for their support.
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References
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The association of VDR gene polymorphism with OA is controversial. The genetic polymorphism studies with VDR and OA are showing both the positive and negative associations (Keen et al., 1997; Uitterlinden et al., 2004; Huang et al., 2000; Baldwin et al., 2002). This inconsistency may also be due to differences in the relative importance of this gene in different races, differences in environmental factors related to vitamin D metabolism, or the presence of other genetic factors that influence VDR function. Further, the association of genetic factors with knee OA is diminishing later in life due to the effects of lifestyle factors, thus it may be difficult to find out their association in the elderly (Muraki et al., 2011). Selection of single SNP, negative statistical significance between cases and controls could be our limitations. In conclusion, the results of our study suggests that genetic variant in FokI does not associate with primary knee osteoarthritis in South Indian population.
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Fig. 1. Representation of correlation between vitamin D levels and VDR genotype.
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Yasovanthi, J., Venkata Karunakar, K., Sri Manjari, K., Pulla Reddy, B., Ajeya Kumar, P., Sesha Charyulu, M., et al., 2011. Association of vitamin D receptor gene polymorphisms with BMD and their effect on 1, 25-dihydroxy vitamin D3 levels in pre- and postmenopausal South Indian women from Andhra Pradesh. Clin. Chim. Acta 412 (7–8), 541–544. Zhu, Z.H., Jin, X.Z., Zhang, W., Chen, M., Ye, D.Q., Zhai, Y., et al., 2014. Associations between vitamin D receptor gene polymorphisms and osteoarthritis: an updated metaanalysis. Rheumatology (Oxford) 53 (6), 998–1008. Zilahi, E., Chen, J.Q., Papp, G., Szántó, A., Zeher, M., 2015. Lack of association of vitamin D receptor gene polymorphisms/haplotypes in Sjögren's syndrome. Clin. Rheumatol. 34 (2), 247–253.
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Uitterlinden, A.G., Burger, H., Huang, Q., Odding, E., Duijn, C.M., Hofman, A., et al., 1997. Vitamin D receptor genotype is associated with radiographic osteoarthritis at the knee. J. Clin. Invest. 100 (2), 259–263. Uitterlinden, A.G., Fang, Y., van Meurs, J.B., van Leeuwen, H., Pols, H.A., 2004. Vitamin D receptor gene polymorphisms in relation to Vitamin D related disease states. J. Steroid Biochem. Mol. Biol. 89–90 (1–5), 187–193. Valdes, A.M., Hart, D.J., Jones, K.A., Surdulescu, G., Swarbrick, P., Doyle, D.V., et al., 2004. Association study of candidate genes for the prevalence and progression of knee osteoarthritis. Arthritis Rheum. 50 (8), 2497–2507. van Schoor, N.M., Zambon, S., Castell, M.V., Cooper, C., Denkinger, M., Dennison, E.M., et al., 2015. Impact of clinical osteoarthritis of the hip, knee and hand on self-rated health in six European countries: the European Project on Osteoarthritis. Qual Life Res (Epub ahead of print). Vasilopoulos, Y., Sarafidou, T., Kotsa, K., Papadimitriou, M., Goutzelas, Y., Stamatis, C., et al., 2013. VDR TaqI is associated with obesity in the Greek population. Gene 512 (2), 237–239.
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Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Contents lists available at ScienceDirect
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Krishna Subramanyam a,1, Subhadra Poornima b,1, Keerthi Konda Juturu b, Dev Anand c, Shruthy Mohanthy d, Imran Ali khan e, Qurratulain Hasan b,⁎
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Article history: Received 26 February 2016 Received in revised form 30 April 2016 Accepted 3 May 2016 Available online xxxx
Department of Orthopedics, Yashoda Hospitals, Hyderabad 500036, India Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad 500068, India Department of Orthopedics, Kamineni Institute of Medical Sciences, Nalgonda 508254, India d Department of Biochemistry, Kamineni Institute of Medical Sciences, Nalgonda 508254, India e Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, P.O. Box-10219, King Saud University, Riyadh 11433, Saudi Arabia
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Osteoarthritis (OA) is a common and multifactorial skeletal disabling disease in the adult/elder population. Vitamin D was involved with multiple diseases including OA. Based on the prior studies, the single nucleotide polymorphism in the vitamin D receptor (VDR) gene might be a risk factor for the development of autoimmune diseases. However, we aimed to investigate whether FokI polymorphism and serum vitamin D levels are in association with primary knee OA in south Indian population. In this case–control study we have selected 100 OA cases and 100 healthy controls from Kamineni Hospitals in Telangana and India. The collected blood samples were used for estimation of serum vitamin D levels and isolation of genomic DNA. PCR was carried out with specific primers. The FokI enzyme was used to perform RFLP and followed by electrophoresis and analysis. The clinical data has been calculated separately between the OA cases and controls. The biochemical analysis performed with ELISA method revealed that 44% of cases were vitamin D deficient, 28% had in sufficient vitamin D levels and 28% had sufficient vitamin D levels. We have performed the statistical analysis between the OA cases and controls and we could not find any significant association either with allele and genotype frequencies (p N 0.05). The gender association analysis also failed to disclose the significant difference between males and females (p N 0.05). The results of our present study concludethat genetic missense variant of FokI in VDR gene does not associate with primary knee OA in the south Indian population. © 2016 Published by Elsevier Inc.
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Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population
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Keywords: Osteoarthritis VDR gene FokI polymorphism Asian Indian population
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1. Introduction
Osteoarthritis (OA) is a common chronic and multifactorial etiology with a group of mechanical abnormalities involving degradation of joints and subchondral bone (He et al., 2014). The prevalence of OA varies widely between studies and depends on the study population and increases with age with osteoarthritic changes uncommon under the age of 40, but seen in most people over the age of 70. Obesity and overweight are the major factors that became rapidly growing threat to worldwide health and the co morbidities include hypertension, type2 diabetes, stroke and cardiovascular diseases.
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Abbreviations: OA, Osteoarthritis; VDR, Vitamin D Receptor; PCR, Polymerase chain reaction; RFLP, Restriction Fragment Length Polymorphism; ELISA, Enzyme Linked Immunosorbent Assay; SNP, Single Nucleotide Polymorphism; SNPs, Single Nucleotide Polymorphisms; K/L Grade, Kellgren/Lawrence Grade; 25OHVD, 25-hydroxyvitamin D; OR, Odds Ratio; CIs, Class Intervals. ⁎ Corresponding author at: Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad 500068, Telangana, India. E-mail address: [email protected] (Q. Hasan). 1 Equal contribution.
The knee, hip and hand are most affected by the disease (Van Schoor et al., 2015). Knee OA is the commonest degenerative arthritis. Knee OA is a mechanically driven disease and it is suggested that medial tibiofemoral knee-joint load increases with pharmacologic pain relief, indicating that it may be positively associated with disease progression. The underlying mechanisms are still unknown; a genetic component has been established by population studies. Genetic factors contribute to the development of OA the heritability has been estimated at 48%– 65% (Ai et al., 2014). The World Health Organization Scientific Group on Rheumatic Diseases estimates that among those people who are 60 or older, the incidence rate of OA is up to 1 in 10 (Pereira et al., 2011). Age, sex, obesity, ethnicity and profession are the multiple risk factors and well recognized environmental factors, genetic variations have also been found to be strong determinants (Cai et al., 2014). The involvement of vitamin D in bone and cartilage metabolisms could explain why alterations in vitamin D homeostasis are associated to several pathological conditions of knee cartilage and intervertebral disc tissue, in particular OA and disc degeneration linked pathologies (Colombini et al., 2013). Single nucleotide polymorphisms (SNPs) are single base variations between genomes, useful for genome-wide
http://dx.doi.org/10.1016/j.genrep.2016.05.001 2452-0144/© 2016 Published by Elsevier Inc.
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Medicine, Kamineni hospitals (Khan et al., 2015). The genomic DNA was quantified with NanoDrop and stored at −80 °C until further use. Genotyping determination for fok1 polymorphism in the VDR gene, located in exon 2 was carried out with polymerase chain reaction and restriction fragment length polymorphism performed with 2% agarose gel electrophoresis.
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2.5. Statistical Analysis
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2. Materials and methods
The association of genotype in OA was examined by odds ratio analysis with 95% confidence interval using Openepi software (Khan et al., 2014). The allele frequencies of VDR gene were tested to determine whether they were in accordance with HWE (Hardy–Weinberg equilibrium). Clinical data are expressed as mean ± standard deviation. Data analysed by ANOVA. An independent t-test was used to test the case and controls. Statistical significance was defined as p b 0.05.
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2.1. Ethics
3. Results
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3.1. Demographic details of the patients and controls
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Ethical approval for this current research was obtained from the Institutional Ethics Committee, Kamineni Hospitals, Hyderabad, India. Data and blood collection from study individuals were collected after taking their informed consent.
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2.2. Patient recruitment
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The hospital based case–control study has been carried out in one hundred (n = 100) south Indian OA patients who were clinically diagnosed and radiologically confirmed with primary knee OA and 100 healthy controls were arbitrarily selected from the routine master health checkup examination who did not have any complaints of OA or autoimmune diseases. The selection, inclusion and exclusion criteria of cases and controls have been published in our earlier paper (Poornima et al., 2015). All the patients were recruited from Department of Orthopedics, Kamineni hospitals, Hyderabad, India. The age range of the patients was 25–60 years (42.4 ± 8.1) and the age range of controls was 26–62 (42.1 ± 7.9). Personal, medical and family histories were documented in a well-designed proforma.
The clinical features of cases and controls have been described in Table 1. The mean age of the cases was 42.4 ± 8.1 and controls was 42.1 ± 7.9, which are similar (p = 0.87). The clinical features such as age, gender and height were also similar between cases and controls. The weight and BMI of cases was significantly greater (p b 0.05). Comorbid factors hypertension, thyroid dysfunction and T2DM were more in the cases compared to controls (p b 0.05). In this study, 28% of the cases had positive family history of OA.
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2.3. OA score
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The radiographic image and clinical information were used for the Kellgren/Lawrence (K/L) score (0–4 scale). K/L system is a method of classifying the severity of knee osteoarthritis using five grades (Kellgren and Lawrence, 1957)
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1. Grade 0: No radiographic features of Osteoarthritis
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2. Grade 1: Doubtful joint space narrowing and possible osteophytic lipping. 3. Grade 2: Definite osteophytes and possible joint space narrowing on anteroposterior weight bearing radiograph 4. Grade 3: Multiple osteophytes, definite joint space narrowing, sclerosispossible bony deformity. 6. Grade 4: Large osteophytes marked joint space narrowing, severe sclerosis and definitely bony deformity.
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2.4. Investigation of FokI polymorphism
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Four milliliters of the peripheral blood was collected and 2 mL of the serum was used for measurement of 25-hydroxyvitamin D (25 OH VD) levels, by ELISA method using IDA kit manufactured by Immunodiagnostic Systems Ltd., UK. Individuals were categorized as deficient (b20 ng/ml), insufficient (20–30 ng/ml), sufficient (30–100 ng/ml) and potential toxic (N100 ng/ml). The remaining 2 mL of the blood was used for genomic DNA extraction using salting out technique, the standard method used in the Department of Genetics and Molecular
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3.2. Biochemical analysis
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mapping and study of disease genes. SNP is a useful marker for diseases in haplotype-based association studies and in linkage disequilibrium analysis (Khan et al., 2016). Ng and Henikoff (2001) have predicted the effect of SNPs on protein function based on sequence homology methods and many polymorphisms in vitamin D Receptor (VDR) were identified. VDR gene covers 105 kb, with an extensive promoter region capable of generating multiple tissue-specific transcripts (Colombini et al., 2013). VDR harbors several known functional polymorphisms and some of these have been investigated (He and Wang, 2015). Several studies investigated the role of VDR (FokI)/rs2228570 polymorphism in the susceptibility to common diseases such as T2DM, osteoporosis and OA. Based on the prior studies, we have carried out our study to investigate whether FokI polymorphism and serum vitamin D levels are in association with south Indian population.
The serum concentration of 25 OH VD was measured using ELISA method. This test was performed for the identification of regulation of bone metabolism and calcium homeostasis effects as a deficiency or excess of vitamin D. We have measured the vitamin D levels in knee osteoarthritic cases. The results of our study showed that 44% of cases were vitamin D deficient, 28% had in sufficient vitamin D levels and 28% had sufficient vitamin D levels.
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3.3. Polymorphism analysis
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The genotype and allele distribution of Fok1 variants in OA cases and controls is shown in Table 2. Genotype distribution deviated from the HWE with total absence of TT genotype in both cases and controls. The genotype distribution in OA cases was 95% in AA and 5% in AT, whereas in controls, the genotypic distribution was 99% in AA and 1% in AT. The allele frequencies between the cases and controls did not showed significant difference i.e. T vs A: OR-5.1 (95% CI: 0.5–44.0); p = 0.13. Both dominant and recessive mode of inheritance did not show significant difference between cases and controls. Statistically we could not find any significance among our cases and controls.
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Table 1 Demographic details of the patients and controls.
t1:1 t1:2
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Characters
Cases (n = 100)
Controls (n = 100)
p value
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Age (years) Sex: (M:F) Height (cm) Weight (kg) BMI (kg/m2) Age of onset Family history of OA History of HTN History of T2DM History of thyroid dysfunction
42.41 ± 8.11 32:68 156.63 ± 2.68 76.97 ± 8.44 31.38 ± 3.4 39.52 ± 6.89 28 (28%) 50 (50%) 38 (38%) 39 (39%)
42.17 ± 7.98 31:69 155.46 ± 2.45 69.78 ± 7.87 25.89 ± 2.67 NA NA 15 (15%) 14 (14%) 22 (22%)
0.87 0.88 0.37 0.001 0.01 NA NA b0.01 b0.01 b0.01
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
K. Subramanyam et al. / Gene Reports xxx (2016) xxx–xxx Table 2 Genotype and allele distribution of VDR-Fok1 gene polymorphism.
t2:3
Genotype
OA cases
Controls
OR 95% CI
t2:4 t2:5 t2:6 t2:7 t2:8
CC CT TT C T
95 (95%) 05(5%) 0 (0%) 195 (0.975) 05 (0.025)
99 (99%) 01 (1%) 0(0%) 199 (0.995) 01 (0.005)
1.00 5.2 (0.5–45.40) 3.1 (0.61–15.87) 1.00 5.1 (0.5–44.5)
p value
Vitamin D levels
CC genotype
CT genotype
TT genotype
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0.13 0.16
Deficient Insufficient Sufficient Toxic
43% 34% 23% 0
60% 20% 20% 0
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t4:4 t4:5 t4:6 t4:7
0.13
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3.4. Statistical correlation between genders
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We tried to correlate the OA cases with the gender subjects, i.e. males (32%) vs. females (68%). The genotype and allele frequency of A25920T variants with gender have been documented in Table 3. Genotypic and allelic distribution did not show any statistical significance between males and females i.e. T vs A: OR-2.1 (95% CI: 0.12–4.78); p = 0.56. (See Table 4.)
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3.5. Correlation between vitamin D levels and VDR genotype in cases
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In this study we found deficiency, insufficiency and sufficiency of vitamin D levels. 43% of the cases were deficient vitamin D levels with AA genotype, 34% had insufficient vitamin D with AA genotype and 23% had sufficient vitamin D levels with homozygous AA genotype. We found 60% cases with AT genotype in which 3 cases had deficient vitamin D, and two cases had sufficient vitamin D levels (Fig 1). When we compared the vitamin D levels of OA cases and degenerative spine arthritis cases with random controls of general population the percentage of individuals who had deficient vitamin D levels was higher (Table 5).
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4. Discussion
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The aim of this study was to identify the association between FokI polymorphism of VDR gene in primary knee OA in Asian Indian population. Our results were displayed as negative association and we could not find the statistical significance between cases and controls. Serum 25-hydroxyvitamin D (25OHD) concentration is an effective indicator of vitamin D status in humans (Jia et al., 2015). Most of the cases exposed the deficiency of vitamin D levels (44%) and 28% of cases were insufficiency and 28% had sufficient vitamin D levels. Deficient levels of vitamin D levels in an individual with a variant allele are associated with the diseases. One of our unpublished data in our cohort showed statistical significance between spine degenerative cases and healthy controls. VDR gene polymorphism may be associated with the smaller joints like spine but not like knee joints. In our study, we found 5 heterozygous CT VDR genotype cases, 4 were obese and 1 were overweight with respect to BMI, which is indicating obesity with heterozygous genotype individuals are more prone to the disease. An evidence that osteoarthritis, diabetes, cancer, cardiovascular diseases, tuberculosis,
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Table 3 Genotype and allele frequencies of VDR-Fok1 gene polymorphism between males and females.
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virus infections, urinary stones, periodontitis, and autoimmune diseases have been reported with FokI polymorphism in VDR gene might be associated with the development of autoimmune diseases (Zilahi et al., 2015). VDR polymorphism, such as ApaI (rs7975232), Taq1 (rs731236), Bsm1 (rs1544410) and FokI (rs2228570) have been reported to be associated with various diseases. FokI located in exon 2, results in different translation initiation sites on VDR gene. This is the only known VDR polymorphism that translates in different protein products (Uitterlinden et al., 2004). Earlier studies reported the association between VDR polymorphisms and obesity (Vasilopoulos et al., 2013) for different ethnic group (Binh et al., 2011) and for type 2 diabetic subjects (Ye et al., 2001). Earlier studies have showed the positive (Vasilopoulos et al., 2013) and negative (Huang et al., 2000; Colombini et al., 2013; Valdes et al., 2004; Uitterlinden et al., 1997) association with VDR gene and OA. FokI genetic variant was positively associated with Broncho pulmonary dysplasia, T2DM, pre/post-menopausal women and falls, balance and muscle power (Koroglu et al., 2014; Al-Daghri et al., 2012; Yasovanthi et al., 2011; Barr et al., 2010). Liu et al. (2014) from their meta-analysis studies concluded that the variants in the VDR gene such as Bsm1, Taq1 and Apa1 might not be an important predictor for OA. Zhu et al. (2014) studies also performed the meta-analysis studies was designed between the Europe versus Asian subjects and their results suggested that VDR gene polymorphism play a role in OA aetiology in the Asian population. Our results were not in accordance with the above supported articles. Bid et al. (2005) had carried out the VDR gene polymorphisms such as FokI, TaqI and ApaI in normal healthy controls and this data has compared with other ethnicity results and concluded that the frequency and distribution of the polymorphisms in India are substantially different from other ethnic population groups. The functional effects of the VDR TaqI polymorphism is located near the 3′-untranslated region i.e. in intron 8/exon 9. Human VDR gene is mapped at chromosome 12q12-q14, and five common polymorphisms have been typically associated with VDR activity, namely VDR Tru9I (rs757343), FokI (rs2228570), TaqI (rs731236), BsmI (rs1544410) and ApaI (rs7975232). Few studies have suggested that these polymorphisms might alter polyadenylation of the VDR mRNA transcript, and thus affect mRNA stability. VDR protein and gene plays an important role in vitamin D pathway and it is an intracellular hormone receptor that specifically binds the biologically active form of calcitriol or vitamin D, 1,25-dihydroxyvitamin D and interacts with specific nucleotide sequences of target genes to produce a variety of biologic effects and involves conversion of serum 25-hydroxyvitamin D into active hormone, 1,25-dihydroxyvitamin D. The protein in VDR is known to display polymorphic variation, belongs to the steroid hormone family of nuclear receptors which are responsible for the transcriptional regulation of a number of hormone responsive genes. (Uitterlinden et al., 2004; He and Wang, 2015).
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However one of our unpublished data revealed that recessive mode of inheritance showed significant association between degenerative spine arthritic cases and controls (4.25 95% CI {1.62–11.14} p = 0.003).
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Table 4 Correlation between vitamin D levels and VDR genotype in OA cases.
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Genotype OA males (n = 32) OA females (n = 68) OR 95% CI
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01(3%) 00 (0%) 63 (0.99) 01 (0.01)
04 (6%) 00 (0%) 132(0.97) 04 (0.03)
2.12 (0.12–35.06) 0.50 (0.05–4.73) 0.83 (0.15–4.49) Reference 2.12 (0.12–4.78)
0.55 0.82 0.56
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Table 5 t5:1 Comparison of vitamin D levels between spine degenerative arthritis cases, osteoarthritis t5:2 cases and random healthy controls. t5:3 Vitamin D levels
Degenerative spine arthritic cases
OA cases
Random healthy controls
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Deficiency Insufficiency Sufficiency Toxic
43% 34% 23% 0
44% 28% 28% 0
52% 29% 19% 0
t5:6 t5:7 t5:8 t5:9
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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All the authors declare that there is no conflict of interests towards this article.
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Acknowledgement
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We are thankful to the patients for their support towards this study and we are thankful to Kamineni hospitals for their support.
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References
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The association of VDR gene polymorphism with OA is controversial. The genetic polymorphism studies with VDR and OA are showing both the positive and negative associations (Keen et al., 1997; Uitterlinden et al., 2004; Huang et al., 2000; Baldwin et al., 2002). This inconsistency may also be due to differences in the relative importance of this gene in different races, differences in environmental factors related to vitamin D metabolism, or the presence of other genetic factors that influence VDR function. Further, the association of genetic factors with knee OA is diminishing later in life due to the effects of lifestyle factors, thus it may be difficult to find out their association in the elderly (Muraki et al., 2011). Selection of single SNP, negative statistical significance between cases and controls could be our limitations. In conclusion, the results of our study suggests that genetic variant in FokI does not associate with primary knee osteoarthritis in South Indian population.
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Fig. 1. Representation of correlation between vitamin D levels and VDR genotype.
Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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Please cite this article as: Subramanyam, K., et al., Missense FokI variant in the vitamin D receptor gene in primary knee osteoarthritis patients in south Indian population, Gene Reports (2016), http://dx.doi.org/10.1016/j.genrep.2016.05.001
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