Vitamin D deficiency and rs731236(Taq1) vitamin D receptor gene polymorphism as possible risk factors for rheumatoid arthritis and osteoarthritis

Abstract

Introduction: Epidemiological evidence indicates a significant association between vitamin D deficiency and an increased
incidence of autoimmune diseases. The role of vitamin D and the vitamin D receptor (VDR) on rheumatic arthritis (RA) and
osteoarthritis (OA) health has recently been placed in the spotlight. The aim of this study is to investigate any association between
circulating vitamin D level and rs731236 (Taq1) vitamin D receptor gene polymorphism among Jordanian Rheumatic arthritis and
osteoarthritis patients.
Methods: this study enrolled one hundred and eighty four RA patients, one hundred and fifty four OA patients and two hundred
healthy Jordanians from Prince Hamzah Hospital/Amman (2013-2015). 25-hydroxyvitamin D (25 (OH) D) level was determined by
competitive immunoassay. DNA was extracted from whole blood for all samples and control, PCR was amplified using specific primers,
rs731236 (Taq1) VDR restriction digestion was carried out for Taq1 genotyping
Results: 25 (OH) D level was statistically lower among RA and OA patients (12.03± 3.21, 11.88 ± 4.63 ng/mL,) compared to
the healthy controls (21.32± 3.12 and 21.51±3.46 ng/mL; respectively). 25 (OH) D within rs731236(Taq1)VDR TT genotype among
RA patients (11.67± 3.24) was statistically lower than its control (21.23± 3.43). Statistical difference was found in 25 (OH) D level
within TT genotype among the OA patients (12.63±4.31 ng/mL) compared to its control (21.41±3.32 ng/mL).
Conclusion: This study showed that there is statistically significant decrease in the mean vitamin D level among both RA and
OA patients compared to control, and rs731236(Taq1)VDR TT genotype may be a possible risk factors for both RA and OA.

Full text

VITAMIN D DEFICIENCY AND RS731236(TAQ1) VITAMIN D RECEPTOR GENE POLYMORPHISM
AS POSSIBLE RISK FACTORS FOR RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
SAMEER AHMED AL HAJ MAHMOUD1, MANAR FAYIZ ATOUM2, HUDA MUSTAFA AL-HOURANI3, SAJEDAH BATEINEH4,
SALIM ABDERRAHMAN5, FOAD ALZOUGHOOL6
1Assistant professor, Faculty of Medicine, The Hashemite University, Zarqa, Jordan - 2Professor, Department of Medical Laboratory
Sciences, Faculty of Allied Health Sciences, Hashemite University, Zarqa, Jordan - 3Associate professor, Clinical Nutrition and Dietetics,
Faculty of Allied Health Sciences, Hashemite University, 4Lecturer, Department of Medical Laboratory Sciences. Faculty of Allied
Health Sciences, Hashemite University, Zarqa, Jordan - 5Assistant professor, Faculty of Science, Department of Biology and
Biotechnology, The Hashemite University - 6Dr Foad Alzoughool, assistant professor, Department of Medical Laboratory Sciences,
Faculty of Allied Health Sciences, Hashemite University, Zarqa, Jordan
Introduction
Vitamin D is a seco-steroid hormone that is
absorbed from sunlight and diet, its metabolism
starts from liver where it is hydroxylated to
25(OH)D, then bond to vitamin D binding protein
in kidneys and converted to 1,25 (OH)2D(1).
Vitamin D is important for many physiological
processes; it plays a role in mineralization home-
ostasis through phosphorous and calcium metabo-
lism. It has an immunoregulatory role that include
the enhancement of microbicide ability of mono-
cytes/macrophages and the down-modulation of
inflammatory cytokines produced by T lympho-
cytes(2). It also decrease MHC-II expression and
increase apoptosis process(3). Its deficiency might
trigger an autoimmune response and might involve
in autoimmune rheumatic diseases(4).
Rheumatic arthritis is a chronic multifactorial,
autoimmune, inflammatory disorder that affects the
Acta Medica Mediterranea, 2018, 34: 209
Received August 30, 2017; Accepted January 20, 2018
ABSTRACT
Introduction: Epidemiological evidence indicates a significant association between vitamin D deficiency and an increased
incidence of autoimmune diseases. The role of vitamin D and the vitamin D receptor (VDR) on rheumatic arthritis (RA) and
osteoarthritis (OA) health has recently been placed in the spotlight. The aim of this study is to investigate any association between
circulating vitamin D level and rs731236 (Taq1) vitamin D receptor gene polymorphism among Jordanian Rheumatic arthritis and
osteoarthritis patients.
Methods: this study enrolled one hundred and eighty four RA patients, one hundred and fifty four OA patients and two hundred
healthy Jordanians from Prince Hamzah Hospital/Amman (2013-2015). 25-hydroxyvitamin D (25 (OH) D) level was determined by
competitive immunoassay. DNA was extracted from whole blood for all samples and control, PCR was amplified using specific pri-
mers, rs731236 (Taq1) VDR restriction digestion was carried out for Taq1 genotyping
Results: 25 (OH) D level was statistically lower among RA and OA patients (12.03± 3.21, 11.88 ± 4.63 ng/mL,) compared to
the healthy controls (21.32± 3.12 and 21.51±3.46 ng/mL; respectively). 25 (OH) D within rs731236(Taq1)VDR TT genotype among
RA patients (11.67± 3.24) was statistically lower than its control (21.23± 3.43). Statistical difference was found in 25 (OH) D level
within TT genotype among the OA patients (12.63±4.31 ng/mL) compared to its control (21.41±3.32 ng/mL).
Conclusion: This study showed that there is statistically significant decrease in the mean vitamin D level among both RA and
OA patients compared to control, and rs731236(Taq1)VDR TT genotype may be a possible risk factors for both RA and OA.
Keywords: osteoarthritis, rheumatoid arthritis, serum 25-hydroxyvitamin D.
DOI: 10.19193/0393-6384_2018_1_34

lining of joints, causing a painful swelling that can
eventually result in bone erosion and joint deformi-
ty. RA is distributed worldwide with an estimated
prevalence of 1 to 2% that increases with age,
approaching 5% in women over the age fifty-five(5).
Vitamin D deficiency plays an important factor in
initial rheumatic disease development and worsen-
ing the disease once it presents. Hypovitaminosis D
is highly prevalent among autoimmune rheumatic
diseases population(6). Vitamin D level correlates
inversely with RA activity and serum vitamin D
level is usually low in patients with RA(7). Vitamin
D supplementation in RA patients with persisting
disease activity and vitamin D deficiency con-
tributed to significant improvement in disease
activity within a short duration(8).
Osteoarthritis is a form of arthritis that is char-
acterized by gradual cartilage loss which eventually
result in functional failure of synovial joints. It is
the most common cause of musculoskeletal disabil-
ity that is characterized by loss of articular cartilage
and change in the bones surrounding the joints.
Vitamin D has many biological effects on cartilage
and bone. Vitamin D deficiency exacerbates pain,
dysfunction and a poorer quality of life in patients
with knee osteoarthritis(9). Vitamin D supplementa-
tion could retard the progression of effusion-syn-
ovitis which can potentially benefit people with an
inflammatory OA phenotype(10). Vitamin D supple-
ments modify OA progression through vitamin D
receptor that are found in human articular chondro-
cytes(11).
Vitamin D acts through VDR and polymor-
phism in VDR modifies its function and its metabo-
lism(12) which varies among various ethnic groups.
Epidemiological studies concerning the association
between vitamin D level, OA, RA and
rs731236(Taq1) gene polymorphism are limited and
up to our knowledge this is the first study among
Jordanian that will determine serum 25-hydroxyvit-
amin D among OA and RA patients and investigate
rs731236(Taq1)VDR gene polymorphism among
them.
Materials and methods
Population
One hundred and eighty four RA, one hundred
and fifty four OA patients and two hundred age
matched healthy control (free of any history of RA
or OA) from the Rheumatic clinic at Prince
Hamzeh Hospital/ Amman (2013-2015) were
enrolled in this study. RA control was age matched
with RA patients, while OA control was age
matched with OA patients. All patients with vitamin
D supplements were excluded from this study. RA
and OA were diagnosed by specialized rheumatolo-
gist and fulfilled Rheumatic arthritis classification
for RA patients and OA classification(13,14) respec-
tively. Ethical approval was obtained from the
Institutional Review Board (IRB) at the Hashemite
University and consent forms were signed by all
participants before patient and control interviewing
and sample collection. All samples were collected
during the summer semester (May-October of
2013-2015).
Blood samples
two blood samples (EDTA and plain) were
collected from all participants. Serum was collected
from the plain tubes and stored at 60ºC for 25-
hydroxyvitamin D analysis. EDTA tubes were used
for DNA extraction and analysis.
Vitamin D determination
25(OH)D serum level was determined accord-
ing to the manufacturer instructions by electro-
chemiluminescence method using Elecsys assay kit
(Roche Diagnostics, France) for Beckman Coulter -
Access/Access 2 Immunoassay System.
DNA extraction and PCR amplification
DNA was extracted by Wizard Genomic DNA
extraction and purification kits (Promega, USA),
amplified (Bio Rad iCycler, USA) using: forward
5'-CAGACCATGGACAGGGAGCAA-3' and
reverse 5'-GCAACTCCTCATGGCTGAGGTCTC-
3’ primers that detect rs731236(Taq1)VDR poly-
morphism SNP (rs731236) within VDR exon 9
gene. PCR amplification was carried out in 50µL
reaction volumes using Go Taq Green Master Mix
and according to the manufacture instructions
(Promega, USA). DNA samples were amplified
using programmed PCR protocol: initial denatura-
tion step at 94˚C for 3 min, followed by 30 cycles
at 94ºC for 45s; 58ºC for 60s; and 72ºC for 90s,
then a reaction is carried out at 73ºC for 5 min.
Amplification products were electrophoresed on
2% agarose gel and stained with ethidium bromide.
SNP (rs731236) Taq1 VDR gene was detected by
restriction enzyme digest using the restriction
endonuclease Taq1 digestion (Promega, USA) at
65°C for two hours. Taq1 TT genotype digestion
produce 494bp and 251bp fragments. Taq1 Tt geno-
210 Sameer Ahmed Al Haj Mahmoud, Manar Fayiz Atoum et Al

type produce 494 bp, 293 bp, 251 bp and 201bp
fragments. Taq1 tt genotype produce 293 bp, 251
bp and 201bp fragments. Digested PCR products
then loaded on 2.5% agarose gels, electrophoresed
and visualized under UV lamb. Gels were pho-
tographed using the IP-010-SD photo documenta-
tion system program (Vilber Lourmat, EEC, UK).
Statistical analysis was carried out using the
Statistical Package for Social Sciences version 17.0
and 20.0 (SPSS Inc., Chicago, IL, USA). Chi-
square test was used to evaluate case-control differ-
ences for TaqI genotype distribution among test and
control groups. A t-test was used to assess the sig-
nificance of difference of mean 25(OH)D levels
between test and control groups. Statistical signifi-
cance was defined as P < 0.05.
Results
Both RA and OA patients showed significantly
lower 25(OH)D compared to their control (Table 1).
The mean 25(OH)D serum level was (12.03±3.21,
11.88±4.63 ng/mL) among RA(n=184) and
OA(n=154), compared to (21.32±3.12, 21.51±3.46
ng/mL) among RA control (n=200) and OA control
(n=200) controls, respectively.
Vitamin D level among TT genotype was sig-
nificantly lower among both RA and OA patients
compared to vitamin D level in the control groups.
The results of this study (Table 2 and 3) showed a
statistical significant difference in the mean
25(OH)D levels among TT genotypes within both
RA (11.67 ± 3. 24) and OA (12.63 ± 4.31) patients
compared to RA and OA controls (21.23 ± 3.43;
21.41 ± 3. 32). None of Tt or tt genotypes among
RA or OA patients showed an effect on vitamin D
level.
Discussion
Vitamin D is natural immune modulators that
regulate cellular growth, apoptosis, and other
immunological functions through its VDR receptor.
VDR gene polymorphism plays a major role in the
pathogenesis of both systemic and organ-specific
diseases. Studying vitamin D level and VDR genet-
ic polymorphisms among RA and QA Jordanians is
an important issue because of the high prevalence
of vitamin D deficiency among Jordanians which
accounts for 76% of Jordanian males and 90% of
Jordanian females (15) and because of the lack in
the genetics and biochemical studies among
Jordanian patients except few studies(16-18). Up to our
knowledge, this is the first study that measure
25(OH) D among RA and OA patients and deter-
mine rs731236(Taq1) gene polymorphism among
RA and OA Jordanian patients.
This study showed significantly lower mean
serum 25(OH) D levels among both RA and OA
compared to control (Table 1). In agreement with
our results, Sabbagh et al(19) found lower levels of
25(OH) D among RA. On the other hand, Glover et
Vitamin D deficiency and rs731236(Taq1) vitamin D receptor gene polymorphism as possible risk factors... 211
Mean‡ ± SEM† Control (n=200)
Mean‡ ± SEM† p -value
RA (n=184) 12.03± 3.21* 21.32± 3.12 0.04*
OA(n=154) 11.88± 4.63* 21.51 ± 3.46 0.02*
Table 1: The mean vitamin D level (ng/mL) among
rheumatic arthritis, osteoarthritis patient and control.
RA: rheumatic arthritis, OA: osteoarthritis. *P < 0.05 is calcu-
lated for RA and OA versus control, †SEM is the standard
error of the mean, ‡ mean vitamin D level unit in ng/mL. t- test
and Chi-square test analysis were performed. p<0.05 values
were considered as statistical significant.
rs731236(Taq1)
genotype
RA (n=184)
‡Mean ± †SEM (n)
Control (n=200)
‡Mean ± †SEM (n) p -value
TT 11.67 ± 3. 24 (73) 21.23 ± 3.43 (87) 0.04*
Tt 12.44 ± 5.76 (87) 19.62 ± 5.61 (81) 0.37*
tt 11.22 ± 5.73 (24) 20.42 ± 4.83 (32) 0.22*
Table 2: Vitamin D level (ng/mL) among both rheumatic
arthritis patients and control stratified by Taq1 genoty-
pes.
RA: rheumatic arthritis, OA: osteoarthritis. *P < 0.05 is cal-
culated for RA and OA versus control, †SEM is the standard
error of the mean, ‡ mean vitamin D level unit in ng/mL. t- test
and Chi-square test analysis were performed. p<0.05 values
were considered as statistical significant.
rs731236(Taq1)
genotype
OA (n=154)
‡Mean ± †SEM (n)
Control (n=200)
‡Mean ± †SEM (n) p -value
TT 12.63 ± 4.31 (51) 21.41 ± 3. 32 (77) 0.03*
Tt 11.82 ± 4.34 (69) 19.82 ± 4.61 (91) 0.22*
tt 11.52± 3.76(34) 20.44 ± 4.83 (32) 0.15*
Table 3: Vitamin D level (ng/mL) among osteoarthritis
patients and control stratified by Taq1 genotypes.
RA: rheumatic arthritis, OA: osteoarthritis. *P < 0.05 is cal-
culated for RA and OA versus control, †SEM is the standard
error of the mean, ‡ mean vitamin D level unit in ng/mL. t- test
and Chi-square test analysis were performed. p<0.05 values
were considered as statistical significant.

al(20) and Cao et al(21) found lower levels among OA
patients compared to healthy control. Low levels of
25(OH) D among RA and OA may highlight the
possible role of vitamin D deficiency in the patho-
genesis and treatment of RA and OA. Particularly,
vitamin D supplementation may have a protective
effect during the onset but not during the chronic
stage of OA in the rat model(22).
Vitamin D has immune-regulatory properties
and VDR polymorphism is associated with
increased risk for bone loss in RA and osteoporosis
pathogenesis .VDR gene is located on chromosome
12 (12q13.11) that has11 exons and contains four
polymorphic regions. One of these is
rs731236(Taq1) polymorphism that is located at the
3’-end near the untranslated region at the restriction
site Taq1(23). Although this polymorphism doesn’t
result in structural VDR protein, it may alter gene
expression(24). Inconsistent with the results reported
by Liu et al(24), this study showed significant associ-
ation between Taq1VDR gene polymorphism and
control among RA and OA patients within TT geno-
type. This inconsistency may be due to many fac-
tors such as sample size, clinical heterogeneity, eth-
nicity and multiple pathological mechanisms of RA
and OA. Previous studies showed that
rs731236(Taq1) TT polymorphism have been asso-
ciated with accelerated generalized bone loss
among RA, and associated with increased risk of
Hashimoto’s thyroiditis among Turkish patients(25).
Vitamin D deficiency is also prevalent among
elderly patients with advanced knee OA scheduled
for total knee replacement. Vitamin D deficiency
exacerbates pain, dysfunction and a poorer quality
of life in patients with kneeOA(9).
Supplementation with vitamin D has been pro-
posed as a mean to induce immune tolerance and
thus prevent the development of autoimmune dis-
eases. Vitamin D supplementation could retard the
progression of effusion-synovitis which can poten-
tially benefit people with an inflammatory OA phe-
notype(10).
In conclusion, rs731236(Taq1)TT genotypes is
a possible risk factors for RA and OA, and further
investigating for this associations is recommended,
taking into account gene-gene, gene-environment
interactions, and other confounding factors.
References
1) Hussien YM, Shehata A, Karam RA, Alzahrani SS,
Magdy H, et al. Polymorphism in vitamin D receptor
and osteoprotegerin genes in Egyptian rheumatoid
arthritis patients with and without osteoporosis. Mol
Biol Rep 2013; 40: 3675-80.
2) Ishikawa LL, Colavite PM, Fraga-Silva TF, Mimura
LA, França TG, et al., Vitamin D Deficiency and
Rheumatoid Arthritis. Clin Rev Allergy Immunol 2017;
52: 373-388.
3) Balato A, Schiattarella M, Lembo S, Mattii M, Prevete
N, et al. Interleukin-1 family members are enhanced in
psoriasis and suppressed by vitamin D and retinoic
acid. Arch Dermatol Res 2013; 305: 255-262. doi:
10.1007/s00403-013-1327-8.
4) Cutolo M, Pizzorni C, Sulli A. Vitamin D endocrine
system involvement in autoimmune rheumatic diseases.
Autoimmun Rev 2011; 11: 84-87.
5) http://www.hopkinsarthritis.org/arthritis-info/rheuma-
toid-arthritis/ra-symptoms (20 march, 2017)
6) Zheng ZH, Gao CC, Wu ZZ, Liu SY, Li TF, et al, High
prevalence of hypovitaminosis D of patients with
autoimmune rheumatic diseases in China. Am J Clin
Exp Immunol 2016; 5: 48-54.
7) Lee YH, Bae SC. Vitamin D level in rheumatoid arthri-
tis and its correlation with the disease activity: a meta-
analysis. Clin Exp Rheumatol 2016; 34: 827-833.
8) Chandrashekara S, Patted A. Role of vitamin D supple-
mentation in improving disease activity in rheumatoid
arthritis: An exploratory study. Int J Rheum Dis 2015;
Oct 20. doi: 10.1111/1756-185X.12770.
9) Alkan G, Akgol G. Do vitamin D levels affect the clini-
cal prognoses of patients with knee osteoarthritis? J.
Back. Musculoskelet Rehabil 2016; Mar 27. doi:
10.3233/BMR-160589.
10) Wang X, Cicuttini F, Jin X, Wluka AE, Han W, et al.
Knee effusion-synovitis volume measurement and
effects of vitamin D supplementation in patients with
knee osteoarthritis. Osteoarthritis Cartilage. 2017 Mar
6. pii: S1063-4584(17)30885-3. doi:
10.1016/j.joca.2017.02.804.
11) Tetlow LC, Woolley DE. Expression of vitamin D
receptors and matrix metalloproteinases in osteoarthrit-
ic cartilage and human articular chondrocytes in vitro.
Osteoarthritis Cartilage. 2001; 9: 423-431.
12) Levin GP, Robinson-Cohen C, de Boer IH, Houston
DK, Lohman K, et al. Genetic variants and associations
of 25-hydroxyvitamin D concentrations with major
clinical outcomes. JAMA 2012; 14: 1898-905.
13) Aletaha D, Neogi T, Silman A. Rheumatoid arthritis
classification criteria. Arth Rheum 2010; 62: 2569-
2581.
14) Barg A, Pagenstert GI, Hügle T, Gloyer M, Wiewiorski
M, et al, Ankle osteoarthritis: etiology, diagnostics, and
classification. Foot Ankle Clin 2013; 18: 411-426.
15) Mallah EM, Hamad MF, Elmanaseer MA, Qinna NA,
Idkaidek NM, et al. Plasma concentrations of 25-
hydroxyvitamin D among Jordanians: effect of biologi-
cal and habitual factors on vitamin D status. BMC
Clinical Pathology. 2011; 11: 8.
16) Atoum MF, AlKateeb D, AlHaj Mahmoud SA. The
Fok1 vitamin D receptor gene polymorphism and
25(OH)D serum level and prostate cancer among
212 Sameer Ahmed Al Haj Mahmoud, Manar Fayiz Atoum et Al

Jordanian men. Asian Pac J Cancer Prev 2015; 16:
2227-2230.
17) Atoum MF, Tchoporyan MN. Association between cir-
culating vitamin D, the Taq1 vitamin D receptor gene
polymorphism and colorectal cancer risk among
Jordanians. Asian Pac J Cancer Prev 2014; 15: 7337-
7341.
18) Atoum MF, Alzoughool F. Reduction in breast cancer
susceptibility due to XbaI gene polymorphism of alpha
estrogen receptor gene in Jordanians. Breast Cancer
(Dove Med Press). 2017; 9: 45-49. doi:
10.2147/BCTT.S125652.
19) Sabbagh Z, Markland J, Vatanparast H. Vitamin D sta-
tus is associated with disease activity among rheuma-
tology outpatients. Nutrients 2013; 26: 2268-2275.
20) Glover TL, Goodin BR, Horgas AL, Kindler LL, King
CD, et al. Vitamin D, race, and experimental pain sen-
sitivity in older adults with knee osteoarthritis. Arthritis
Rheum 2012; 64: 3926-3935.
21) Cao Y, Winzenberg T, Nguo K, Lin J, Jones G, et al.
Association between serum levels of 25-hydroxyvita-
min D and osteoarthritis: a systematic review.
Rheumatology (Oxford) 2013; 52: 1323-34.
22) Castillo EC, Hernandez-Cueto MA, Vega-Lopez MA,
Lavalle C, Kouri JB, et al. Effects of Vitamin D
Supplementation during the induction and progression
of osteoarthritis in a rat model. Evid. Based
Complement Alternat Med 2012; 156563. doi:
10.1155/2012/156563.
23) Hustmyer FG, DeLuca HF, Peacock M. ApaI, BsmI,
EcoRI and TaqI polymorphisms at the human vitamin
D receptor gene locus in Caucasians, blacks and
Asians. Hum Mol Genet 1993; 2: 487.
24) Liu H, He H, Li S, Yang L, Wang P, et al. Vitamin D
receptor gene polymorphisms and risk of osteoarthritis:
A meta-analysis. Exp Biol Med (Maywood) 2014; 239:
559-67
25) Yazici D, Yavuz D, Tarcin O, Sancak S, Deyneli O, et
al. Vitamin D receptor gene ApaI, TaqI, FokI and BsmI
polymorphisms in a group of Turkish patients with
Hashimoto’s thyroiditis. Minerva Endocrinol 2013;
38:195-201.
All this work is supported and carried out at the Hashemite
University, Zarqa, Jordan Grant number: (27/2011)
Acknowledgement:
acknowledgments of financial to the Hashemite University and
technical help for the staff at the Health Sciences Department
at the Hashemite University
_________
Corresponding Author:
Dr MANAR ATOUM
E mail: manar@hu.edu.jo
Department of Medical Laboratory Sciences
Faculty of Allied Health, Hashemite University
Zarqa
(Jordan)
Vitamin D deficiency and rs731236(Taq1) vitamin D receptor gene polymorphism as possible risk factors... 213