Original Article

The Association Between Vitamin D Receptor Gene FokI

Polymorphism and Pulmonary Tuberculosis

Dina Octafrida Marpaung1, Bintang Yinke Magdalena Sinaga1, Parluhutan Siagian1,

Erna Mutiara2
1
Department of Pulmonology and Respiratory Medicine, Faculty of Medicine
Universitas Sumatera Utara, Medan
2
Faculty of Public Health Universitas Sumaterwa Utara, Medan

Corresponding Author: Abstract

Dina Octafrida Marpaung | Departement of
Pulmonology and Respiratory Medicine, Backgrounds: There is a wide range of individual responses to
Faculty of Medicine Universitas Sumatera mycobacterium infection. However, the reason why certain people
Utara, Medan| [email protected] suffers from a disease when they are infected with mycobacterium
while others remain healthy is still unknown. Genetic susceptibility
Submitted: July 16th, 2021 is thought to be one of the important explanatory factors for
Accepted: September 23th, 2021 individuals with the risk of developing pulmonary tuberculosis (TB).
Published: October 15th, 2021 FokI polymorphism was one of the genetic variations within the
vitamin D receptor (VDR) gene associated with pulmonary TB. In
the present study, we investigated the association between VDR
Respir Sci. 2021; 2(1): 1-7
https://doi.org/10.36497/respirsci.v1i3.29 gene FokI polymorphism and pulmonary TB.
Method: This is a case-control study with 65 pulmonary
tuberculosis patients as cases and 65 healthy subjects as controls.
The PCR-RFLP technique was used to assess the polymorphism of
the vitamin D receptor gene FokI. The association of the vitamin D
receptor gene FokI polymorphism with pulmonary tuberculosis was
statistically analyzed.
Results: The frequency of FokI genotypes determined from
pulmonary TB patients were 41,5% for FF, 44,6% for Ff and 13,8%
for ff, while in healthy controls, the frequency of FF, Ff and ff were
present in a percentage of; 43,1%, 44,6% and 12,3%, respectively.
Conclusion: There was no significant association between the
VDR gene FokI polymorphism and pulmonary TB.
Keywords: FokI polymorphism, pulmonary TB, vitamin D receptor
gene

INTRODUCTION world, around 10%.1 Approximately one

third of the world’s population has been
Tuberculosis (TB) is the second
infected with Mycobacterium tuberculosis
leading cause of death from infectious
(M. tuberculosis), but only 10% of the
diseases worldwide.1,2 The global number
infected developed into an active
of pulmonary TB cases has been
pulmonary TB disease. The susceptibility to
increasing, compared to previous years.
the disease after the infection of
Indonesia ranks second in the largest
mycobacterium is influenced by the agent,
number of pulmonary TB cases in the
environmental, and genetic factors.2,3

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Dina Octafrida Marpaung, et al

In the past few years, more studies (No: 351/TGL/KEPK FK USU-RSUP

addressing the impact of VDR HAM/2016), that compared risk factors
polymorphisms on TB susceptibility have with disease incidence by comparing a
been conducted in different populations. group of people with pulmonary
Polymorphisms in the VDR gene may tuberculosis (cases) to a group of people
influence VDR activity and subsequent without the disease (control). The sample
downstream vitamin D-mediated effects. 4
data was collected at Haji Adam Malik
Four single nucleotide polymorphisms General Hospital Medan and several other
(SNP), which are the variants of VDR, health centres in Medan, conducted from
including ApaI, BsmI, FokI, and TaqI, are April to June 2016.
the primary focus which have a role in the The sample size in this study was 65
risk of pulmonary TB compared to the other people in the case group and 65 people in
SNPs. 5–7
the control group. The inclusion criteria of
FokI polymorphism (rs2228570) is a case samples in this study were: new
transition polymorphism C to T (ACG to untreated pulmonary TB patients or those
ATG) located in exon 2. FokI who had received antituberculosis
polymorphism, described as the f allele, treatment <7 days, with positive smear
allows the protein translation to start from bacteria in sputum based on direct smear
the first ATG, while the F allele starts the examination or positive GeneXpert
protein translation from the location of the examination results, male and female
second ATG.5–8 Long vitamin D receptors, patients with pulmonary TB aged 18-65
coded from the f allele forms, have more years and agreeing to participate in the
three amino acids, and 1.7 times less study declared in writing (informed
efficient than VDR coded by the F allele consent). Exclusion criteria in this study, on
forms. Therefore, this variant may affect the other hand, included having HIV,
the individuals’ susceptibility to pulmonary Diabetes Mellitus (DM), and other severe
TB.9–11 conditions, such as a history of renal
Based on the above description, the disease or liver disease.
researchers conducted a study to All subjects were given informed
investigate the association betweeen VDR consent, interviewed, and peripheral blood
gene FokI polymorphism with pulmonary samples were obtained. DNA isolation
TB. (Promega, USA) was performed on the
blood and then it was stored at -20°C. The
METHOD genotype analysis of FokI polymorphism of
This was a case control study the VDR gene was performed using

conducted with approval from the Research Polymerase Chain Reaction Restriction
Ethics Committee of the Faculty of Fragment Length Polymorphisms (PCR-

Medicine, University of Sumatera Utara RFLP) technique with endonuclease FokI

restriction enzyme (Thermo Scientific). The

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 The Association Between Vitamin D Receptor Gene FokI Polymorphism and Pulmonary Tuberculosis

primary FokI sequence in this study is heterozygote occurs if there are three
Forward: 5’-AGC TGG CCC TGG CAC TGA bands of 265 bp, 169 bp, and 96 bp.
CTC TGC TCT-3’, while the primary The statistical analysis used in this
Reverse: 5’-ATG GAA ACA CCT TGC TTC research was the logistic regression test.
TTC TCC CTC-3’. Products digested by the The Hardy Weinberg Equilibrium was
restriction enzymes were then visualized in tested on the case group and the control
a Gel Documentation System tool. group using the HWE Testing Calculator
The resulting genotype depends on web tool, which was available online.
the patterns of digestion. The depiction of
electrophoresis, if truncated, would reveal RESULTS

the DNA bands at 169 bp and 96 bp. The characteristics of the case group
Otherwise, the DNA bands would be at 265 and the control group can be seen in Table
bp. The existence of restriction area is 1. Distribution of genotypes and alleles of
marked with the lowercase “f”, while the FokI polymorphism of the VDR gene can be
absence of restriction area is marked with seen in Table 2. The Ff genotype was the
the letter “F”. Whereas FF homozygote highest genotype in both groups, with 29
produces 265 bp band and ff homozygote people (44.6%) in each the case group and
produces 169 bp and 96 bp band, Ff the control group.

Table 1. Characteristic from pulmonary TB patients and healthy people

Case Control
Characteristic
(pulmonary TB) (healthy people)
Age
18-25 years 14 (21.5%) 30 (46.2%)
26-35 years 28 (43.1%) 23 (35.4%)
36-45 years 8 (12.3%) 10 (15.4%)
46-55 years 13 (20.0%) 1 (1.5%)
55-65 years 2 (3.1%) 1 (1.5%)
Gender
Men 47 (72.3%) 42 (64.6%)
Women 18 (27.7%) 23 (35.4%)
Total 65 (100.0%) 65 (100.0%)

Table 2. The frequency of allele and genotype VDR gene FokI polymorphism
FokI Case Control HWE Case HWE Control
P OR (95% CI)
Polymorphism (pulmonary TB) (healthy people) χ2 (p) χ2 (p)
Genotype
FF 27 (41.5%) 28 (43.1%) - 1
0,07 0,01
Ff 29 (44.6%) 29 (44.6%) 0.923 1,037 (0.496-2.169)
(>0.05) (<0.05)
ff 9 (13.8%) 8 (12.3%) 0.781 1,167 (0.393-3.467)
Allele
F 83 (63.8%) 85 (65.4%)
0.795 1.070 (0.643-1.779) - -
f 47 (36.2%) 45 (34.6%)

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Dina Octafrida Marpaung, et al

M UC TB17 TB18 TB19 TB20 TB21 TB22

1500 bp

500 bp

300 bp
265 bp
200 bp 169 bp
100 bp
96 bp

Figure 1. Image of RFLP FokI polymorphism with various genotype in samples from pulmonary TB group
samples. M = marker, UC = uncut. FF genotype in sample TB21 has 1 band (265 bp), ff genotype in samples
TB17 and TB19 have 2 bands (169 bp and 96 bp), Ff genotype in samples TB18, TB20 and TB22 have 3
bands (265 bp, 169 bp, and 96 bp).

There was no statistically significant The PCR product was truncated by

difference in the genotype distribution of the endonuclease FokI restriction enzyme
FokI polymorphism of the VDR gene in the case group can be seen in Figure 1.
between the case group and the control
group (P>0.05). The frequency of the F DISCUSSION
alleles was greater than the frequency of
Individual reactions to
the f alleles in both case group and control
mycobacterium infection vary greatly;
group.
nonetheless, the reason why some people
The F alleles found in the case group
become unwell when infected with
were 83 (63.8%), whereas the f alleles
mycobacterium while others remain
found in the case group were 47 (36.2%).
healthy is unknown. Genetic susceptibility
There was no statistically significant
is thought to be one of the important
difference in the frequency of the F alleles
explanatory factors for individuals with the
and the f alleles in the FokI polymorphism
risk of developing pulmonary TB.13
of the VDR gene between the case group
The VDR gene comprises of a
and the control group (P>0.05). The
complex of intron/exon structures, located
genotype of FokI polymorphism in the case
in the long arm of chromosome 12q12-q14
group was in the Hardy-Weinberg
and consists of 16 exons.5 The VDR gene
equilibrium (P>0.05), whereas the control
is known to be responsible for the genetic
group was not in a state of Hardy-Weinberg
polymorphism affecting receptor activity
equilibrium (P<0.05).12
and vitamin D-mediated effects. 13

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 The Association Between Vitamin D Receptor Gene FokI Polymorphism and Pulmonary Tuberculosis

There are variations in the VDR gene polymorphism of the VDR gene and the
polymorphisms which play a role in the susceptibility of pulmonary TB.3
susceptibility of pulmonary TB, one of This was not in line with a study
which is the FokI polymorphism of the VDR conducted on native Paraguayans which
gene studied in this study. FokI is one of showed a link between the FokI
the VDR variants located in exon 2.14 polymorphism of the VDR gene and
Polymorphism is a variation of DNA pulmonary TB. The meta-analysis study
sequences which causes genetic diversity shows that polymorphisms may have
in the gene pool of a population. different roles in different populations. For
Polymorphism is formed through a example, there was a significant
mutation process which can occur due to relationship between pulmonary TB and
substitution, deletion, or insertion in the the FokI polymorphism of the VDR gene in
order of polynucleotides. Polymorphism Asian population, but it was not found in
has a neutral effect on biological function. African or South American population.4,16,17
However, in some conditions, it may cause The significant relationship between
an impaired biological function. This the FokI and TaqI genotypes of the VDR
happens because there is a change in the gene and the susceptibility to pulmonary
DNA sequences that encode proteins. TB was found in Gambia and London
Polymorphism can also be found in DNA populations.2
areas which do not encode protein.13 Based on a population meta-analysis
In this study, there was no study in China, the variants of homozygote
statistically significant difference in the VDR genes to the FokI polymorphism may
genotype distribution of FokI be more susceptible to pulmonary TB,
polymorphism of the VDR gene between compared to the heterozygote and wild
the case group and the control group. This type variants.9
result was similar to research conducted on The existence of different results
Peru’s population.9 Other studies in from previous studies may be related to
Cambodia, West Africa, and Korea different genetic backgrounds in different
suggested that there was no relationship populations. There are other four SNPs as
between VDR gene polymorphism and variants of VDR, namely ApaI, BsmI, FokI,
pulmonary TB.9,15,16 and TaqI, which play a role in the risk of
Similarly, in the population of pulmonary TB.5–7
Kazakhs, the FokI and ApaI polymorphisms This study only investigated the FokI
did not have a significant relationship with polymorphism of the VDR gene, while there
the risk of pulmonary TB.7 Studies are other SNPs variants of VDR which play
conducted in Indonesia’s population, a role in the risk of pulmonary TB.
especially Batak ethnic, showed that there
was no relationship between the FokI

Respir Sci 2021, Vol.2 No.1 5

Dina Octafrida Marpaung, et al

CONCLUSION 6. Huang L, Liu C, Liao G, Yang X, Tang

X, Chen J. Vitamin D Receptor Gene
There was no significant association
FokI Polymorphism Contributes to
between VDR gene FokI polymorphism and
Increasing the Risk of Tuberculosis: An
pulmonary TB.
Update Meta-Analysis. Medicine
(Baltimore). 2015;94(51).
REFERENCES
7. Zhabagin M, Abilova Z, Askapuli A, et
1. World Health Organization. Global al. Vitamin D Receptor Gene

Tuberculosis Report 2015, 20th Ed. Polymorphisms in Susceptibility to

Geneva; 2015. Tuberculosis in the Kazakh Population

2. Wu L, H D, Y Z, et al. An association in Almaty and Almaty Area. Cent Asian

study of NRAMP1, VDR, MBL and their J Glob Heal. 2014;2(Suppl).

interaction with the susceptibility to 8. Salimi S, Farajian-Mashhadi F, Alavi-

tuberculosis in a Chinese population. Naini R, Talebian G, Narooie-Nejad M.

Int J Infect Dis. 2015;38:129-135. Association between vitamin D

3. Sinaga BYM, Amin M, Siregar Y, receptor polymorphisms and

Sarumpaet SM. Correlation between haplotypes with pulmonary

Vitamin D receptor gene FOKI and tuberculosis. Biomed reports.
BSMI polymorphisms and the 2015;3(2):189-194.

susceptibility to pulmonary 9. Chen C, Liu Q, Zhu L, Yang H, Lu W.

tuberculosis in an Indonesian Batak- Vitamin D receptor gene

ethnic population - PubMed. Acta Med polymorphisms on the risk of

Indones. 2014;46(4):275-282. tuberculosis, a meta-analysis of 29

4. Gao L, Tao Y, Zhang L, Jin Q. Vitamin case-control studies. PLoS One.
D receptor genetic polymorphisms and 2013;8(12).
tuberculosis: updated systematic 10. Larcombe L, Mookherjee N, Slater J, et
review and meta-analysis - PubMed. al. Vitamin D in a Northern Canadian

Int J Tuberc Lung Dis. 2010;14(1):15- First Nation Population: Dietary

23. Intake, Serum Concentrations and

5. Tanabe R, Kawamura Y, Tsugawa N, Functional Gene Polymorphisms. PLoS

et al. Effects of Fok-I polymorphism in One. 2012;7(11):e49872.

vitamin D receptor gene on serum 25- 11. Rashedi J, Asgharzadeh M, Moaddab

hydroxyvitamin D, bone-specific SR, et al. Vitamin D Receptor Gene

alkaline phosphatase and calcaneal Polymorphism and Vitamin D Plasma
quantitative ultrasound parameters in Concentration: Correlation with

young adults. Asia Pac J Clin Nutr. Susceptibility to Tuberculosis. Adv

2015;24(2):329-335. Pharm Bull. 2014;4(Suppl 2):607.

6 https://respiratoryscience.or.id/
 The Association Between Vitamin D Receptor Gene FokI Polymorphism and Pulmonary Tuberculosis

12. Software: hardy-weinberg.

http://www.genes.urg.uk/software/ha
rdy-weinberg.shtml. Published 2017.
Accessed July 15, 2017.
13. Mahmoud AA, Ali AHK. Vitamin D
receptor gene polymorphism and 25
hydroxy vitamin D levels in Egyptian
patients with pulmonary tuberculosis.
Egypt J Chest Dis Tuberc.
2014;63(3):651-655.
14. Thaha I, Yusuf I, Hadju V, Massi N.
Polimorfisme ApaI, FokI, BsmI Gen
Reseptor Vitamin D terhadap Kejadian
Multidrug Resisten Tuberkulosis
(MDR-TB). JST Kesehat.
2012;2(4):390-397.
15. Joshi L, Ponnana M, Penmetsa S,
Nallari P, Valluri V, Gaddam S. Serum
vitamin D levels and VDR
polymorphisms (BsmI and FokI) in
patients and their household contacts
susceptible to tuberculosis. Scand J
Immunol. 2014;79(2):113-119.
16. Cao Y, Wang X, Cao Z, Cheng X.
Vitamin D receptor gene FokI
polymorphisms and tuberculosis
susceptibility: a meta-analysis. Arch
Med Sci. 2016;12(5):1118-1134.
17. Kang T, Jin S, Yeum C, et al. Vitamin
D Receptor Gene TaqI, BsmI and FokI
Polymorphisms in Korean Patients with
Tuberculosis. Immune Netw.
2011;11(5):253.

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