Manganese Superoxide Dismutase Gene Ala16Val Polymorphism in

Pulmonary Tuberculosis Patients with Diabetes at Balai Pengobatan

Lung Disease, Medan City

Mutiara Indah Sari

, Milahayati Daulay

and Dian Dwi Wahyuni

Department of Biochemistry, Faculty of Medicine, Universitas Sumatera Utara, Jl.Dr. Mansur No.5 Medan, Indonesia

Department of Physiology, Faculty of Medicine, Universitas Sumatera Utara, Jl.Dr. Mansur No.5 Medan, Indonesia

Department of Microbiology, Faculty of Medicine, Universitas Sumatera Utara, Jl.Dr. Mansur No.5 Medan, Indonesia

Keywords: Mn-SOD, Polymorphism, Tuberculosis, Diabetes.

Abstract: Manganese Superoxide dismutase (Mn-SOD) gene Ala16Val polymorphism has been shown to be associated

with several infectious disease and metabolic disorder. This study to analyze Mn-SOD gene Ala16Val

polymorphism in patients with pulmonary tuberculosis (PTB) and also suffer from diabetes mellitus (DM).

The study was conducted at Balai Pengobatan Lung Disease, Medan and Integrated Laboratory of Faculty of

Medicine, University of Sumatera Utara (USU), Medan. Mn-SOD gene Ala16Val polymorphism was studied

in 40 outpatient at Balai Pengobatan Lung Diseases, Medan City. The blood glucose was measured with a

spectrophotometer, using commercial glucose kits. Polymerase chain reaction-restriction fragment lengths

polymorphisms (PCR-RFLP) technique was done with the BsaW1 restriction enzyme. SPSS version 22 was

used for statistical data. Ala and Val were analyzed to see the frequency of genotype of Mn-SOD gene

16Ala/Val polymorphism by direct counting. Hardy-Weinberg Equilibrium was analyzed by a Chi-square

test. The results show that the percentage of Val/Val genotype (57.5%) of Mn-SOD gene was higher compared

Ala/Val and Ala/Ala genotypes (37.5%, 5 %) in PTB patients with diabetes. This preliminary study results

indicated that Val/Val genotype was common genotypes in the Mn-SOD gene in PTB patients with diabetes

at Balai Pengobatan Lung, Medan city.

1 INTRODUCTION

Since the beginning of the 20th century, clinicians

have observed an association between pulmonary

tuberculosis (PTB) disease and diabetes mellitus

(DM), although it is still difficult to determine

whether PTB leads to clinical manifestations of

diabetes or diabetes preceding PTB. PTB and

diabetes often co-exist, especially in high-risk

populations for PTB infection (Dooley and Chaisson,

2009). Diabetes can increase the severity and

frequency of a disease, including PTB infection

(Podell, 2014). Indonesia as a country that ranked

fourth in the number of patients with most PTB also

ranked fourth in the number of diabetics (WHO,

2016).

Superoxide Dismutase (SOD) is an enzymatic

antioxidant and classified as a major antioxidant for

the defense of body cells against oxidative stress.

Mann and Kleilin first identified SOD antioxidant at

the 1938 year. SOD has a role in preventing free

radicals and oxidative stress. SOD consists of

intracellular, mitochondrial, and extracellular

enzymes also referred to as type 1 SOD (Cu Zn-

SOD), type 2 SOD (Mn-SOD) and type 3 (EC-SOD)

respectively. SOD levels used as parameters of

antioxidant status in the body and bio-indicator of

oxidative stress conditions (Weisiger and Fridovich,

1973).

Research by Saad et al. (2016) shows an

association of EC-SOD gene polymorphism in DM

(Saad, et al., 2016). Some studies have found the role

of manganese SOD (Mn-SOD) gene Ala16Val

polymorphism with diabetes (Pourvali, et al., 2016).

Mn-SOD is SOD antioxidant in the mitochondria.

Mn-SOD gene is located on chromosome 6q25. The

gene polymorphism of this antioxidants was

substitution alanine (Ala) to valine (Val). It has been

reported that the Mn-SOD Val/Val genotype has

lower activity than Ala/Ala genotype (Bresciani, et

al., 2013). So better diabetes control was found only

in patients with Ala/Ala genotype (Pacal, et al.,

600

Sari, M., Daulay, M. and Wahyuni, D.

Manganese Superoxide Dismutase Gene Ala16Val Polymorphism in Pulmonary Tuberculosis Patients with Diabetes at Balai Pengobatan Lung Disease, Medan City.

DOI: 10.5220/0010080106000604

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

600-604

ISBN: 978-989-758-449-7

2011). Research of association of PTB with

antioxidant gene polymorphism show by

Yuniastuti'research et al. (2017) was founded the

presence of antioxidant gene polymorphism in PTB

patients in Semarang city (Yuniastuti, et al., 2017).

Based on the results of previous research, the study of

Mn-SOD gene Ala16val polymorphism in PTB

patients and Mn-SOD gene Ala16val polymorphism

in diabetic patients has been done before. But

research to find out how Mn-SOD gene Ala16val

polymorphism in PTB patients with diabetes has

never been done. The current study would like to see

how the distribution of Mn-SOD gene Ala16Val

polymorphism in patients with PTB and also suffer

from diabetes at Balai Pengobatan Lung Diseases, in

Medan city.

2 METHOD

This research was performed from February to June

2018 in an outpatient at Balai Pengobatan Lung

Diseases, Medan city. The study has received

approval from the ethics committee of the Faculty of

Medicine, University of Sumatera Utara (USU) with

the letter of ethical approval No. 327/ FK USU.

Forty of PTB patients who also suffer from DM

were diagnosed according to the criteria of

Indonesian Lung Doctor Association (PDPI, 2011)

and standards of Indonesian Endocrinology Society

(PERKENI, 2015) as subjects in this research. The

subjects requested to fill out and sign an informed

consent after being explained the purpose and

benefits of the study. The subjects who 1) took the

antioxidants drugs before the examination, 2) had co-

infections such as HIV, hepatitis, malignant disease

3) had a positive family history of diabetes mellitus

were excluded from this research

Data collection of characteristics subjects: age,

gender and duration of illness were done through

interviews using questionnaires. The blood sample

were taken three ml from the mediana cubital vein,

using the EDTA vacuum syringe. Then, the blood

was centrifuged for 10 minutes at a rate of 3000 rpm.

The blood glucose was measured with a

spectrophotometer at a wavelength of 500 nm, using

commercial glucose kits. Analysed of Mn-SOD gene

was performed on blood samples of subjects at

Integrated Laboratory of Faculty of Medicine,

University of Sumatera Utara (USU). DNA for

genomics was extracted from whole blood, using the

genomic DNA extraction kit from Promega. DNA

was amplified by PCR amplification method, using

Forward primer 5'-CAG CCC AGC CTG CGG AGA

CGG-3 'and Reverse primer 5'-CTT GGC CAA CGC

CTC CTG GTA CTT-3'. Before performing the

amplified process, PCR mix was created first with 25

µl PCR reaction volume consists of 12.5 µl

GoTaq

Green Master Mix, one µl Forward primer, 1

µl Reverse primer, 2 µl DNA template, 8.5 µl

nuclease free water (Promega, USA).

The PCR conditions involved at 95 °C for 5 min for

an initial denaturation of DNA, followed with (95 °C

for 45 s (melting), 54 °C for 30 s (annealing) and

72 °C for 30 s) x 30, and a final extension at 72 °C for

5 min. PCR products were analyzed by 2% agarose

gel electrophoresis in staining with ethidium bromide.

Then, 10 µl of the PCR reaction mixture was digested

by the 0.2 µl of restriction endonuclease BsaW1

enzyme at 60 C in 10 minutes with restriction

fragment length polymorphism (RFLP) technique.

The RFLP product was visualized by electrophoresis

technique at 4% agarose gel. The cutting point of Mn-

SOD gene by restriction enzyme results in a 267-bp

product (Ala16/ala) or a 267 bp, 183 bp and 84 bp

(Ala16val) or 183 and 84 bp products (Val16val)

(Souiden, et al., 2016). SPSS version 22 was used for

statistical data. Ala and Val were analyzed to see

frequencies of genotypes of Mn-SOD gene Ala16val

polymorphism by direct counting. Hardy-Weinberg

Equilibrium (HWE) was analyzed by a Chi-square

test.

3 RESULT AND DISCUSSION

This study was done on 40 of the PTB patients with

diabetes. The characteristics of the subjects in this

study are shown in Table 1.

Table 1: Distribution of subject’s characteristics

Characteristics

N=40 (%)

Mean, SD

Age (years)

53.26±8.77

Gender (male/

female)

26(65)/14(35)

Duration of disease

(years)

3.61±1.38

Blood glucose

levels (mg/dl)

295.51±57.8

Table 1 shows the mean values of the age of PTB

patients with diabetes was 53.26 years (SD = 8.77).

Age is the risk factors for suffering diseases, such as

infectious disease and metabolic disorder. The

condition is probably caused by some changes in

body composition which is due to the aging process.

Manganese Superoxide Dismutase Gene Ala16Val Polymorphism in Pulmonary Tuberculosis Patients with Diabetes at Balai Pengobatan

Lung Disease, Medan City

601

Aging causes a decrease in the immune system and

the function of the organs of a body (Rochmah, 2009).

In the present research, based on gender found

that male in subjects group more than female (65%

VS 35%). Gender is not a risk factor for infectious

disease, especially PTB infection, but gender may be

a risk factor for diabetes. Females are more likely to

have diabetes than males (Mihardja, 2009). The

proportion of the female in form as central obesity

results in a decrease in the action of insulin in the

target tissue. It will be leading to an increase in

getting diabetes (Flier and Maratos-Flier, 2010). The

mean value of the duration of disease was for 3.61

years. The mean level of BGLs in this study subjects

was 295.51 mg/dl. The length duration of DM will

cause the parameters of BGLs will get worse (Sari

et al., 2018). The high level of BGLs showed that DM

disease in this group still not well controlled

The amplified PCR products of the Mn-SOD gene

gave a 267 bp fragment. PCR is a method for

detecting gene molecules of samples from blood,

urine, saliva, etc. PCR is an enzymatic method for

DNA amplification in vitro. PCR products can be

identified using agarose gel electrophoresis.

Currently, a PCR-based molecular method has been

designed to analyze gene polymorphisms. One of the

ways that can be used to find out genotypes of the

Mn-SOD gene polymorphism is the PCR-Restriction

Fragment Length Polymorphism" (PCR-RFLP)

(Rodwell, et al., 2015)

In this research, the product of RFLP-PCR of Mn-

SOD gene that digested by the BsaW1enzyme can be

seen in Figure 1.

Figure 1: PCR-RFLP product of Mn-SOD gene Ala16val

polymorphism on 4% agarose gel electrophoresis.

In Figure 1, it can be seen that after the digestion

with BsaW1 restriction enzyme, homozygous wild-

type Ala/Ala showed up at bands 267 bp;

heterozygous Ala/Val showed three bands at 267 bp,

183 bp, and 84 bp; and homozygous mutant Val/Val

showed two bands at 183 bp and 84 bp.

Restriction enzymes are catalyst compounds that

can cut the gene on certain positions at separate

locations. This cut will be resulting in some fragment

of DNA (Rodwell, et al., 2015).

In this forty subjects of PTB patients with diabetes,

the distribution of genotype of Mn-SOD gene

Ala16Val polymorphism can be seen in Table 2

Table 2: The frequency of genotypes within SOD gene

Ala16Val polymorphism

Genotype

(%)

Ala/Ala

Ala/Val

37.50

Val/Val

57.50

Total

100

At the Table 2 can be seen, the frequency of

Val/Val genotype was higher than the Ala/Val and

Ala /Ala genotype (57.50% VS 37.50% VS 5%). The

results are similar to the previous study by Nakanishi

et al. which determines Val/Val genotype is higher

than the Ala /Val and Ala /Ala genotype (Nakanishi,

et al., 2008). Previous research has found an increase

in the frequency of Ala/Val genotype compared to

Ala/ala and Val/Val genotypes in the Mn-SOD gene

polymorphism of DM patients in the Lebanese

population (Zahreddine, et al., 2018).

SOD is an antioxidant as a major defense against

oxidative stress that induced by increased reactive

oxygen species (ROS). Oxidative stress plays an

important role in the pathogenesis of some diseases

such as infectious disease, metabolic disorder, etc. In

PTB with diabetic patients happen an abnormality in

immunity and cell-mediated phagocyte function. The

process will produce ROS which will increase

oxidant and lipid peroxides. Previous research has

shown an increase in lipid peroxides compounds such

as malondialdehyde in DM (Sari

, et al., 2018). The

oxidative stress causes increasing the need for SOD

antioxidants. MnSOD, a homotetramer containing

one manganese ion per subunit. Specific Mn-SOD

gene sequences genetically express Mn-SOD. The

Mn-SOD gene is located on chromosome six. If there

is a polymorphism in the Mn-SOD gene, it will

probably affect the expression level of the Mn-SOD

gene (Bresciani, et al., 2013).

Polymorphism is a change of nucleotide

sequences in genes that do not cause changes in

protein structure but result in variations in protein

Lane M : DNA Ladder 25 bp

Lane 1,2,4 : homozygous mutant Val/Val

Lane 3,6 : heterozygous Ala/Val

Lane 5 : homozygous Ala/Ala

4 5

267 bp

183 bp

84 bp

25 bp

300 bp

50 bp

75 bp

100 bp

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

602

function. Polymorphism can determine susceptibility

to disease. The impact of polymorphism is a change

in the vulnerability of a population to illness. In this

study, the Mn-SOD gene Ala16Val polymorphism of

PTB patients with diabetes showed that the Val/Val

genotype frequency was higher compared the wild-

type Ala/Ala. Ala/Ala genotype was wild-type of Mn-

SOD gene and the occurrence of variations such as

Ala/Val heterezygote and Val/Val mutant-

homozygote may occur is due to adaptation to the

environment that is inherited genetically to next

generation (Williams, et al., 1998). Usually, the

presence of polymorphism relates only to a person's

susceptibility to disease but in this present results

have not been able to prove that these genetic

variations contribute to the risk of vulnerability to

suffer PTB with diabetes. Genotype analysis is

required in the healthy control group as a comparison.

Hardy-Weinberg Equilibrium was analysis

performed by the chi-square test. In this research, the

Mn-SOD gene Ala16val polymorphisms in PTB

patients with diabetes is consistent with the HWE (p>

0.05). The relative proportions of genotype in the

population of PTB patients with diabetes constant

from one generation to the next. The genotype in this

population still in equilibrium is due to the possibility

of random marriage, and no migration to the

population (Andrews, 2010).

4 CONCLUSION

In this research shows that the Val/Val genotype of

Mn-SOD gene polymorphism has a higher percentage

than Ala/Val and wild-type homozygote Ala/Ala

genotype. It is necessary to conduct a study that

compares the group of PTB patients with diabetes

with the healthy control group to see the genotype

mutant as a risk factor for the development of a

disease.

ACKNOWLEDGEMENT

The present research is supported by the University

of Sumatera Utara (USU) under the research grant

TALENTA USU of the Year 2018 with Contract

Number 2590/UN5.1.R/PPM/2018.

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