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Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy Venkata Sanjeev Kumar Neela a, Naveen Chandra Suryadevara a, Vidya Gouri Shinde a, Satya Sudheer Pydi a, Suman Jain b, Surya Satyanarayana Singh c, Vijaya Lakshmi Valluri a, M.P.J.S. Anandaraj a,⇑ a b c

LEPRA India – Blue Peter Public Health & Research Centre (BPHRC), Hyderabad, Telangana, India Thalassemia and Sickle Cell Anaemia Society (TSCS), Hyderabad, AP, India Department of Biochemistry, Osmania University, Hyderabad, AP, India

a r t i c l e

i n f o

Article history: Received 9 August 2013 Revised 2 April 2015 Accepted 9 April 2015 Available online xxxx Keywords: Vitamin D Receptor Leprosy Polymorphisms Haplotype

a b s t r a c t Background: Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive immune responses through its ligand and polymorphisms in VDR gene may affect its regulatory function. Objective: To investigate the association of three VDR gene polymorphisms (TaqI rs731236, FokI rs2228570 and ApaI rs7975232) with leprosy. Methods: The study group includes 404 participants of which 222 were leprosy patients (paucibacillary = 87, multibacillary = 135) and 182 healthy controls. Genotyping was done using PCR-RFLP technique. Statistical analysis was performed using SNP Stats and PLINK software. Results: The VDR FokI (rs2228570) ff genotype, ApaI (rs7975232) AA, Aa genotype and haplotype T-f-a, T-F-A were positively associated with leprosy when compared to healthy controls. Conclusion: The two variants at Fok and Apa positions in VDR gene are significantly associated with leprosy. Genotypes at FokI (ff), ApaI (aa) and haplotype (T-F-a, T-f-a) may contribute to the risk of developing leprosy by altering VDR phenotype/levels subsequently modulation of immune response. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

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1. Introduction

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Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae and still remains as a serious public health problem, despite being at the stage of elimination leprosy is prevalent in certain endemic pockets of countries such as India, Brazil, Indonesia, Philippines, Madagascar, Myanmar, Nepal and Mozambique as reported. The global prevalence of leprosy at the beginning of 2012 is 181,941 cases as reported to World Health Organization (WHO) and amongst them India represents 58% of new cases detected. Leprosy is a spectral disease exhibiting two extreme poles involving tuberculoid form with a strong cell mediated response and lepromatous form with low cell mediated response often with Th2 response, and a transition between the

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Abbreviations: PB, paucibacillary leprosy; MB, multibacillary leprosy; RFLP, Restriction Fragment Length Polymorphism; VDR, Vitamin D Receptor. ⇑ Corresponding author.

clinical forms. The clinical outcome is determined by the immunological response mounted by the host [1]. The lepromatous leprosy and Borderline lepromatous are classified as multibacillary leprosy (MB) while tuberculoid leprosy and borderline tuberculoid are classified as paucibacillary leprosy (PB) according to WHO norms. Host immune system plays an important role in controlling and combating the infection. Genetic defects/polymorphisms in these immune responsive genes results in making an individual more prone to infections. Genetic association studies have indicated that HLA [2] and several non-HLA genes influence either leprosy susceptibility or the clinical form of leprosy that develops upon infection [3,4]. One such gene is Vitamin D Receptor (VDR) which is a member of the nuclear receptor supergene family and trans-acting transcriptional regulatory factor. The ligand Vitamin D mediates many immuno-modulatory effects through VDR. VDR is found on many immune cells, such as macrophages, dendritic cells, T and B lymphocytes, mainly after activation [5] and plays a vital role in the innate immune response [6]. Polymorphisms in VDR gene

http://dx.doi.org/10.1016/j.humimm.2015.04.002 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.

Please cite this article in press as: Neela VSK et al. Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy. Hum Immunol (2015), http://dx.doi.org/10.1016/j.humimm.2015.04.002

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have been associated with metabolic, auto immune and infectious diseases such as leprosy [7,8], tuberculosis [9], Crohn’s disease [10], Leishmaniasis [11], Parkinson disease [12], asthma [13], and cancer [14]. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of mycobacterium and this effect may be influenced by polymorphisms in the VDR gene. The three variants Taq, Fok and Apa in VDR gene are prominently associated with various diseases while the genotype and allelic association is still inconsistent between different cohorts with leprosy disease [7,8,15]. In VDR gene the Fok1 polymorphism is near the 50 -UTR region of the gene within the DNA-binding domain and Taq I and ApaI polymorphisms are close to the 30 -UTR region. The UTR regions play an essential role in message stability and post transcriptional processes. The Fok polymorphism is functional, since the F allele results in short 424 aa VDR while f allele results in a longer 427 aa VDR protein. Uitterlinden et al. demonstrated that the 424 aa VDR variant is fairly more active than the 427 aa variant in terms of its transactivation capacity, as a transcription factor [16]. Additionally Evelyne et al. by transfection experiments reported that the presence of the short F-VDR results in a higher NFAT- and NF-kB-driven transcription capacity than in the presence of the long f-VDR [17]. There are very few reports explaining the association of these three polymorphisms and no reports explaining the haplotype association in leprosy. Hence the study aims to identify the association of Taq, Fok and Apa polymorphisms with PB and MB leprosy.

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2. Subjects and methods

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2.1. Study subjects

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The study includes 404 participants, who were prospectively recruited at the clinic of LEPRA India, Blue Peter Public Health and Research Centre (BPHRC) of which 222 were leprosy patients and 182 age and sex matched healthy controls from the same geographical area (Hyderabad, Andhra Pradesh, India) (Suppl Table 1). HIV seropositivity and familial history of leprosy and tuberculosis were considered as exclusion criteria while selecting the healthy controls. Patients were classified into PB (n = 87) and MB leprosy (n = 135) groups as per WHO classification, patients showing negative smears at all sites are grouped as paucibacillary leprosy (PB), while those showing positive smears at any site were classified as multibacillary leprosy (MB) (http://www.who.int/lep/classification/en/index.html). Peripheral blood was collected from all the participants only after obtaining informed and written consent. The study was approved by the Institutional Ethics Committee.

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2.2. DNA isolation

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DNA was extracted from venous blood using Flexigene DNA kit according to the manufacturer instructions (QIAGEN, Hilden, Germany). DNA concentrations were estimated using ND-1000 spectrophotometer (Thermoscientifics, Wilmington DE, USA).

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2.3. Single nucleotide polymorphisms

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Genotyping of VDR Taq, Fok and Apa polymorphisms was performed by PCR-RFLP (Restriction Fragment Length Polymorphism) method using specific restriction enzymes.

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2.3.1. Polymerase chain reaction The primer sequences for the Fok [18], Taq and Apa [7] polymorphisms were used as reported earlier. PCR was performed using optimum conditions standardised. PCR products were

visualised under UV and photographed with Image lab software. (BIO RAD, XR+, USA).

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2.3.2. Restriction Fragment Length Polymorphism (RFLP) Restriction digestion was performed by digesting PCR products with respective restriction enzymes (NEB). Taq I (catalogue # R0149) restriction enzyme at 65 °C for 3 h, Fok I (catalogue # R0109) enzyme at 37 °C for 3 h and Apa I (catalogue # R0114) enzyme at 25 °C overnight and the digested reaction mixture was then resolved on 2.5% Agarose gel and the gels were visualised under UV by gel documentation system (BIO RAD, XR+, USA). The digested allele was designated with lower case (t, f and a) letter and undigested allele with upper case letter (T, F and A) for Taq, Fok and Apa positions respectively. 50 amplified PCR products including both leprosy patients and healthy controls were outsourced (Eurofins India, Bangalore) for sequencing to validate RFLP genotype results.

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2.4. Statistical analysis

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The overall genotype frequencies were compared by 3  2 Chisquare (v2) test to determine genotype association. A ‘p’ value of 0.80) between was observed (data not shown).

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Please cite this article in press as: Neela VSK et al. Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy. Hum Immunol (2015), http://dx.doi.org/10.1016/j.humimm.2015.04.002

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V.S.K. Neela et al. / Human Immunology xxx (2015) xxx–xxx Table 1 Allele and genotype frequencies of VDR Taq I, Fok I & Apa I polymorphisms in leprosy patients and healthy controls. TaqI (rs731236)

Leprosy per se (222)

MB (135)

PB (87)

HC (182)

p-Value of v2 test for overall genotypic frequencya,b,c

TT Tt tt TT vs Tt + tt tt vs Tt + TT FokI (rs2228570) FF Ff

95 (42.8) 104 (46.8) 23 (10.4)

55 (40.7) 68 (50.4) 12 (8.9)

40 (46) 36 (41.4) 11 (12.6)

68 (37.4) 83 (45.6) 31 (17)

0.12, 0.11, 0.35

121 (54.5) 80 (36)

82 (60.7) 42 (31.1)

39 (44.8) 38 (43.7)

100 (54.9) 74 (40.7)

0.12, 0.12, 0.05

ff

21 (9.5)

11 (8.1)

10 (11.5)

8 (4.4)

p-Value of v2 test for individual genotypic frequencyd,e,f

Reference 0.12, 0.09, 0.05 0.93, 0.25, 0.08

FF vs Ff + ff

0.04, 0.15, 0.03

ff vs Ff + FF

0.34, 0.05, 0.54

ApaI (rs7975232) AA Aa

77 (34.7) 106 (47.7)

42 (31.1) 69 (51.1)

35 (40.2) 37 (42.5)

92 (50.5) 74 (40.7)

aa

39 (17.6)

24 (17.8)

15 (17.2)

16 (8.8)

0.001, 0.001, 0.08

Reference 0.001, 0.0008, 0.38 0.001, 0.0005, 0.19

AA vs Aa + aa

0.008, 0.01, 0.36

aa vs Aa + AA

0.15, 0.07, 0.53

OR (95% CI)g,h,i

0.89 1.38 2.17 3.33 1.02 1.57 2.27 2.87

(0.59–1.35), (0.80–2.37) (0.92–5.10), (1.21–9.13) (0.69–1.51), (0.93–2.64) (0.98–5.26), (1.08–7.61)

1.71 0.74 2.92 0.61 1.93 0.71 2.23 0.71

(1.12–2.61), (0.43–1.30) (1.51–5.63), (0.28–1.35) (1.29–2.88), (0.42–1.19) (1.20–4.15), (0.34–1.49)

0.67 (0.41–1.08), 1.70 (0.65–4.44), 0.77 (0.49–1.21), 1.98 (0.77–5.07),

2.03 (1.24–3.32), 3.39 (1.63–7.06), 2.27 (1.42–3.62), 2.32 (1.18–4.59),

p-Value for 3  2 v2 test of comparison of overall genotype frequencies between aleprosy per se and healthy controls, bmultibacillary (MB) patients and healthy controls, paucibacillary (PB) patients and healthy controls. d,g p-Value and OR for comparison of frequencies between leprosy per se and healthy controls by logistic regression. e,h p-Value and OR for comparison of frequencies between multibacillary (MB) patients and healthy controls by logistic regression. f,i p-Value and OR for comparison of frequencies between paucibacillary (PB) patients and healthy controls by logistic regression. c

Table 2 Significant haplotype in PB. MB and leprosy per se groups.

Per se

MB PB

Haplotype

Frequency

Chi square

OR

p value

T-f-a T-F-a T-F-A T-F-a T-F-A T-f-a

0.083 0.245 0.217 0.246 0.219 0.085

5.01 10.6 5.51 13.7 5.57 6.51

2.06 1.85 0.65 2.21 0.603 2.57

0.025 0.001 0.01 0.0002 0.01 0.01

p-Value calculated by Chi-square and value

Association of Taq I, Fok I and Apa I polymorphisms in Vitamin D Receptor (VDR) gene with leprosy.

Vitamin D Receptor (VDR) is a transacting transcription factor which mediates immunomodulatory function and plays a key role in innate and adaptive im...
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