Polymorphism of Vitamin D Fok1 Receptor Gene in Patients with

Pulmonary Tuberculosis of Batak Ethnic

Debby Mirani Lubis,

Department of Physiology, Muhammadiyah University of North Sumatera, Gedung Arca No. 53, Medan, Indonesia

Keywords: Vitamin D polymorphism, Fok1 polymorphism, Fok1 Tuberculosis

Abstract: Tuberculosis (TB) is one of the infectious diseases that is still a health problem of the world, especially in

developing countries. The polymorphism of the vitamin D receptor gene (VDR) by some studies may affect

the workings of vitamin D. The polymorphism of this receptor gene causes a person to become more

susceptible to M. tuberculosis infection. This study aimed to determine polymorphism of Vitamin D Fok1

Receptor Gene in Patients with Pulmonary Tuberculosis of Batak Ethnic. A total of 42 patients who met the

inclusion and exclusion criteria were examined for vitamin D Fok1 receptor gene with PCR-RFLP and

analyzed by electrophoresis and the level of vitamin D was examined by ELISA. The results showed that 26

patients had Ff genotype (61,9%), 14 patients have FF genotype (33,3%), and two patients had ff genotype

(4,8%). The most common level of Vitamin D in FF genotype was optimal (64,3%), in Ff genotype was

optimal (53,8%), and ff genotype had the same percentatage of optimal and insufficiency level. Conclusion:

Based on this research, the most common polymorphism of vitamin D Fok1 receptor gene in Batak ethnic is

Ff genotype.

1 INTRODUCTION

TB disease is caused by infection with the bacteria

Mycobacterium tuberculosis (M. tuberculosis),

which was first discovered by Robert Koch in 1903

so that the first disease called Koch Pulmonum

(Chocano-Bedoya & Ronnenberg, 2009). Judging

from the year of the discovery of that germs, it can

be said to be very ironic, because up to now this

disease cannot be eradicated from all over the world,

even new problems arise such as the emergence of

cases of drug-resistant TBC (Multi Drug Resistance)

and TBC cases that accompanied HIV (Human

Immunodeficiency Virus).

Several studies have suggested that there is a

relationship between vitamin D levels and resistance

to TBC. Vitamin D can increase the synthesis of the

innate immune system components through the

Vitamin D receptor (VDR) complex with the active

form of vitamin D (1.25D3), one of which is

cathelicidin which has an important role in fighting

infection from Mycobacterium (Sutaria et al., 2014).

Vitamin D can work when binding to VDR.

The polymorphism of this VDR gene causes a

person to become more susceptible to M.

tuberculosis infection. Some of the vitamin D

vitamin receptor polymorphisms that have been

identified are TaqI, ApaI, BsmI, and FokI.

The polymorphism of the Fok1 VDR gene is

formed by the transition of C to T (ACG-ATG) at

the first and second translational initiation sites in

exon 2. If the translation starts from the first ATG

(individual T allele, written f), the VDR protein

synthesis has a maximum length (427 amino acids).

Conversely, if translation begins at the second ATG

site (individuals with C allele, written as F), then the

VDR protein deficits three amino acids at the

terminal. The shortening of these three amino acids

leads to shorter VDR proteins to be more

functionally active (Chocano-Bedoya &

Ronnenberg, 2009).

Some studies of these polymorphisms have

shown quite varied results, whereas ethnicity also

affects the types of polymorphisms associated with

TB infection. Studies in Turkey showed that only

BsmI variations that have an effect on susceptibility

to TB, this result are different from studies in West

African populations who reportted on variations of

ApaI that are significantly associated with TB,

whereas in Asian populations, the ff genotype of

Fok1 is related to TB, even in a South American

study it was found that none of these polymorphism

Lubis, D.

Polymorphism of Vitamin D Fok1 Receptor Gene in Patients with Pulmonary Tuberculosis of Batak Ethnic.

DOI: 10.5220/0008790600490052

In Proceedings of the 2nd Syiah Kuala International Conference on Medicine and Health Sciences (SKIC-MHS 2018), pages 49-52

ISBN: 978-989-758-438-1

types were significantly associated with TB

(Khalilullah et al., 2014). This suggests that further

study of vitamin D polymorphisms in certain ethnic

groups is warranted.

2 METHOD

This research is analytic research with cross-

sectional design. After getting ethical clearance, the

Subjects were collected at Helvetia, Amplas,

Teladan and Johor Health Center in Medan in

January 2016. The inclusion criteria of subjects were

patients (male or female) with Pulmonary TB

(category 1) aged 18 to 65 years old, with ethnic

Batak obtained from 2 previous generations

(grandparents, father-mother). Patients with immune

deficiencies such as HIV (examined with HIV rapid

test), Diabetes Mellitus (examined with a glucotest

device), history of organ transplants, impaired renal

function, impaired liver function, malignancy,

treatment with steroids, pregnancy and lactation,

extrapulmonary TB patients, taking vitamin D, and

Body Mass Index ≤ 18.5 were excluded. After

signed informed consent, blood samples were taken

as much as 3 cc for examined of FokI gene

polymorphism and vitamin D levels. The blood

samples were centrifuged directly then taken to an

integrated laboratory of Faculty of Medicine,

University of North Sumatera.

The polymorphism examined using PCR-RFLP

and analyzed with electrophoresis gel. On each gel is

given marker, positive control, and negative control.

The result of the tape was visualized on a UV

illuminator device. The resulting genotype depends

on the pattern of digestion. Homozygous FF for the

absence of a digested FokI side with a band of 265

bp; homozygous ff for a perfectly digested Fok1 into

196 bp and 69 bp and heterozygous Ff bands if there

are three bands (265 bp, 196 bp, and 69 bp).

The Levels of vitamin D (25OH) were examined

by ELISA kit (DIAsource®). The absorbance was

read at a wavelength of 450 nm.

3 RESULT

Subjects were 42 people; 27 men (62,5% and 32,5%

respectively). Subjects with the youngest age in this

study was 19 years old and the oldest 63 years old.

3.1. The Levels of Vitamin D

The vitamin D status is classified according to 3

levels; sufficiency (optimal) (30-100 ng/ml),

insufficiency (10-29 ng/ml) and deficiency (<10

ng/ml) (suggested reference values for adults from

ELISA kit brochure). There were 24 subjects

(57,1%) with optimal level, 15 subjects (35,7%)

with insufficiency and three subjects (7,1%) with

deficiency level.

3.2. The Polymorphism of VDR gen Fok1

The results showed that 26 patients have Ff

genotype (61,9%), 14 patients have FF genotype

(33,3%) and two patients have ff genotype (4,8%).

Figure 1: Analyzed bands with electrophoresis gel.

3.3. The Polymorphism and Levels of

Vitamin D

The most common level of Vitamin D in FF

genotype was optimal (64,3%), in Ff genotype was

optimal (53,8%), and ff genotype had the same

percentage of optimal and insufficiency level.

Table 1: The polymorphism and levels of vitamin D

Polymorp

hism

Optimal

(n)

Insufficie

ncy(n)

Deficiency

(n)

4 DISCUSSION

The majority of epidemiologic studies found vitamin

D status less susceptible to tuberculosis but different

from this study where the majority of TB patients

had optimal vitamin D status. These different results

are likely to be influenced by many factors, one of

which is the ethnic factor which from several

previous studies can show different results in

different ethnic populations (Rashedi et al 2015,

Salahuddin et al, 2013; Siswanto et al, 2009; Sutaria

et al 2014)

SKIC-MHS 2018 - The 2nd Syiah Kuala International Conference on Medicine and Health Sciences

In the distribution of Fok1 VDR gene

polymorphism, the most common genotype was

heterozygous (Ff) whereas homozygous ff subjects

were found in 2 patients. The same is true of

research conducted by Sinaga et al. in Indonesia on

Batak ethnic (Sinaga et al., 2014) and Sharma et al.

in India in Chhattisgarth ethnic (Sharma et al.,

2011). However, these results are different from

those of other studies conducted in West Africa

(Bornman et al., 2004), India (Selvaraj et al, 2003),

South Africa (Babb et al, 2007), England (Martineu

et al, 2011) and Iran (Rashedi et al, 2015), which

shows the most commonly encountered genotype is

the FF type. A meta-analysis by Gao et al. (2010)

which states that the ff genotype has risk

susceptibility to TB that is clearly different from the

results obtained in this study because the genotype ff

only found in 2 subjects. Ethnic factors are likely to

affect the distribution of polymorphism genotypes in

each population.

Table 2: Comparison of FokI genotype in TB Patients

from Various Population Studies.

Researcher

(year)

Country

(ethnic)

FF(%)

Ff(%)

ff(%)

Selvaraj

(2003)

India

Bornman

(2004)

West Africa

33,2

4,8

Babb

(2007)

South Africa

Martineu

(2011)

England

Sharma

(2011)

India/

(Chattisgarth)

0,07

Sinaga

(2014)

Indonesia

(Batak)

35,5

55,3

9,2

Rashedi

(2015)

Iran

52,4

39,3

8,3

In terms of theory, subjects with an allele F

should be stronger than the f alleles, so subjects with

ff genotype would be more susceptible to

tuberculosis, but this is not the case in this study

because the genotype ff is very few and the genotype

that has the F allele is susceptible also to

tuberculosis. The possibility of Batak tribe is a few

who have ff genotype and no previous studies are

sufficient to compare the frequency of this genotype.

In addition to this study, there is only one more

study that also studied polymorphism of VDR FokI

gene on Batak tribe that is research by Sinaga et al.

(2014) which also shows a proportion distribution

similar to this study.

5 CONCLUSIONS

Most subjects showed the optimal status of vitamin

D levels. The polymorphisms of Fok1 VDR gene in

Lung TB patients of Batak ethnic encountered in

this study are the heterozygous Ff genotype and the

least of which is the ff genotype. Ethnic factors may

affect a person's susceptibility and correlation to

tuberculosis, so it is necessary to conduct similar

research on different ethnicities with a large number

of samples from this study.

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