Document Type : Original Article
Authors
- Fathia Meirina 1, 2
- Dina Keumala Sari 3
- Inke Nadia Diniyanti Lubis 2
- Rini Savitri Daulay 2
- Finny Fitry Yani 4
- Bugis Mardina Lubis 2
- Rosita Juwita Sembiring 5
- Pandiaman Pandia 6
- Muhammad Rusda 1, 7
- Mustafa Mahmud Amin 1, 8
1 Philosophy Doctor in Medicine Program, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
2 Department of Pediatrics, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
3 Department of Nutrition, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
4 Department of Pediatrics, Faculty of Medicine, Universitas Andalas, Padang, Indonesia
5 Department of Clinical Pathology, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
6 Department of Pulmonology & Respiratory, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
7 Department of Obstetrics & Gynecology, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
8 Department of Psychiatry, Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia
Abstract
Background: Vitamin D levels are affected by several vitamin D metabolism gene. The genetic variations of vitamin D metabolism gene, such as the CYP gene, plays a role in the first and the second vitamin D hydroxylation pathway, in which this will affect the levels of vitamin D serum. Low levels of vitamin D can play a role in the risk development of chronic infection diseases, and even malignancy; one of the aforementioned infections is the M. tuberculosis infection, so that on pregnant with LTBI are at a higher risk of developing into an active TB infection, especially if they are experiencing vitamin D deficiency.
Methods: The design used in this study is a case control study that follows 84 pregnant mothers on their third trimester from three hospitals in Medan, North Sumatera. The subject of this study is 42 pregnant mothers with LTBI and 42 pregnant mothers without LTBI. This study was conducted throughout December 2021 until July 2022. The assessed parameters are the IGRA and the vitamin D levels on pregnant mothers, which then will be further correlated with the CYP2R1 rs10741657 and CYP27B1 rs10877012 gene polymorphism. Further analysis utilized the Chi-squared test and the Fisher test.
Results: This study reported that 42,85% pregnant mothers with LTBI experienced vitamin D deficiency. This study also reported that there is not a significant correlation between CYP2R1 rs10741657 and CYP27B1 rs10877012 gene polymorphism and vitamin D levels (p = 0.541 and 0.057), and there is not a signification correlation between CYP2R1 rs10741657 and CYP27B1 rs10877012 gene polymorphism and the incidence of LTBI (p = 0.03; p = 0.001).
Conclusions: There is no signification correlation between CYP2R1 rs10741657 and CYP27B1 rs10877012 gene polymorphism on the incidence of LTBI and the vitamin D levels on pregnant with LTBI.
Graphical Abstract
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Keywords
Introduction
Latent tuberculosis infection has affected around 900 million women throughout the world [1], where it is estimated that there are as much as 216,500 cases of latent TB infection per year [2]. Latent TB infection during pregnancy has a high risk of developing into an active TB infection. This risk development relies on the immune system of the body [3] and the immune system mechanism is affected by the vitamin D levels. Low vitamin D levels can be found on people who are infected with M. tuberculosis, where a prior meta-analysis study reported that children who are infected with TB has a lower vitamin D levels compared to healthy children [4], and the deficiency of vitamin D is correlated with LTBI progressivity into becoming TB [5]. Meanwhile, deficiency, or even insufficiency of vitamin D can be found in around 1 billion people throughout the world [6, 7] and in Indonesia, around 95% out of 148 adult women suffers from deficiency and insufficiency of vitamin D [6].
Genetic variance on the genes involved in the vitamin D metabolism will affect the levels and function of vitamin D in the body. The decrease in 25(OG)D serum can occur, despite only having a genetic mutation on one base [8]. A study by Genome-wide Association Studies (GWAS) reported that the genes playing a role with the vitamin D metabolism are GC (vitamin D binding protein), CYP24A1, CYP2R1, and DHCR7 in relation to the vitamin D levels and Vitamin D Receptor (VDR), CYP27A1, and CYP27B1, which correlations to the vitamin D metabolic pathway. The study from GWAS reported that GC, CYP2R1, DHCR7, and VDR are correlated with low vitamin D levels [9].
Genetic variance on vitamin D metabolism gene, aside from affecting the levels of vitamin D serum, can also play a role in the risk development of a chronic infection, and even malignancy. In a systematic review study, it was reported that CYP2R1 rs10741657 and CYP27B1 rs10877012 polymorphism has a significant correlation with vitamin D [10, 11]. Other research has also reported that CYP2R1 and CYP27B1 polymorphism, which are the metabolic enzyme of vitamin D, can affect the vitamin D levels, and play a role in chronic diseases such as TB, and even malignancy [12, 13].
Materials and Methods
Study design
This was a case-control study. The participants of this study were 84 pregnant mothers (42 case groups and 42 control groups). The subjects were categorized based on their diagnosis of LTBI or non-LTBI. The collected data included IGRA testing results, vitamin D levels, and CYP2R1 rs10741657 and CYP27B1 rs10877012 polymorphism genes from pregnant mothers in their third trimester of pregnancy, starting from December 2021 until July 2022.
The subjects for this study originated from three hospitals in Medan, North Sumatera, where the subjects were pregnant mothers who routinely underwent prenatal check-ups in research hospitals. This study was conducted directly on the subjects after they agreed to participate and signed an informed consent form. Prior to sign the informed consent form, the participants were given an oral explanation.
Participant
Sampling in this study used consecutive sampling method, where subjects who met the inclusion and exclusion criteria would be selected as research subjects. The inclusion criteria were pregnant women in the third trimester of pregnancy without complications. Pregnant women with pregnancy complications (hypertension, eclampsia, and DM) and pregnant women diagnosed with COVID-19 were included as exclusion criteria. All subjects in this study received vitamin D supplements of 1000 IU daily for one month from their obstetricians.
Statistical analysis
The chi square test or fisher exact test was used to analyze the correlation between CYP2R1 rs10741657 and CYP27B1 rs10877012 gene polymorphisms and vitamin D and ILTB levels (IGRA positive). The significance value was below 0.05 (p < 0.05).
Examination
All examinations for this study were carried out at the Integrated Laboratory of the Faculty of Medicine, University of North Sumatra. Each measurement was performed twice, and the obtained data was the average of measurement results. Blood samples from pregnant women were obtained through the medial cubital vein in the left or right arm, during the third trimester of pregnancy before delivery. Blood samples from the newborn's umbilical cord were taken at the time of delivery. IGRA testing was performed to categorize the case and control groups by Enzyme-linked Immunosorbent Spot Assay (ELISPOT) by assessing the number of spots that appeared on the incubated specimen. The results were read under microscopic view, and if there were £ 5 spots that appeared, it was classified as IGRA-negative, and ³ 6 spots that appeared were classified as IGRA-positive. Vitamin D testing was performed by assessing serum vitamin D levels in mothers and infants by ELISA test, using an Enzyme Linked Analysis Techniques Immunosorbent Assay (ELISA) kit (DiaSorin, USA), with classification of vitamin D (25-hydroxy vitamin D) (25(OH)D3) levels in pregnant women as follows: Normal Maternal Level: > 30 ng/mL, Insufficient Maternal Level: 21-29 ng/mL, and Insufficient Maternal Level: <20 ng/mL.
The CYP2R1 rs10741657 polymorphism gene testing was conducted on pregnant mothers. The initial step is the DNA isolation from the venous blood with the forward primer CYP2R1 5’-GGGAAGAGCAATGACATGGA-3’ and the reverse primer CYP2R1 5’-GCCCTGGAAGACTCATTTTG-3. The CYP2R1 PCR cycle is initial denaturation at 94 ºC for 5 minutes, denaturation at 94 ºC for 30 seconds, annealing at 57 ºC for 30 seconds, extension at 72 ºC for 40 seconds, and the final extension at 72 ºC for 7 minutes. On the CYP27B1 rs10877012 testing, the DNA isolation were done using the forward primer CYP27B1 5’-GCCTGTAGTGCCTTGAGAGG-3’ and the reverse primer CYP27B1 5’-CAGTGGGGAATGAGGGAGT-3’. The CYP27B1 PCR cycle is initial denaturation at 95 ºC for 5 minutes, denaturation at 95 ºC for 30 seconds, annealing at 60 ºC for 30 seconds, extension at 72 ºC for 60 seconds, and final extension at 72 ºC for 10 minutes. The next step is to conduct the PCR restriction length polymorphism (RFLP) testing, which utilized the MN1l enzyme as the restriction enzyme for CYP2R1 rs10741657, with the electrophoresis pattern showing homozygous GG genotype (151bp, 105bp,32 bp), AA (256bp, 32bp), and heterozygous GA (256 bp, 151bp, 105bp, 32bp). On the PCR RFLP for the CYP27B1 rs10877012 gene, the HINFL enzyme was utilized as the restriction enzyme, with the electrophoresis pattern showing homozygous GG genotype (138bp, 49bp), TT (187bp), and heterozygous GT (187bp, 138bp, 49bp).
Ethics approval and consent to participate
This study received ethical approval based on the Declaration of Helsinki guidelines. All research subjects involved read and were given an explanation of the study. All research subjects understood and signed the consent. All research procedures have been ethically tested and approved by the Ethics Committee of the University of North Sumatra, no. 1012/KEPK/USU/2021, dated 8 October 2021.
Results and Discussion
From the 84 participants, 42 pregnant mothers without LTBI (IGRA-negative) were categorized as the control group and 42 pregnant mothers with LTBI (IGRA-positive) were categorized as the case group (Figure 1).
The subjects for this study are pregnant mothers in their third trimester of pregnancy without any pregnancy complication that routinely did antenatal check-ups in research hospitals. In this study, the number of adult pregnant mothers were 82 people, and young adult pregnant mothers were 2 people. The largest amounts of pregnant mothers that were of Javanese descent consisted of 28 out of 84 people. 47 pregnant mothers worked as a housewife, 16 people worked as an employee at a state-owned company (SOEs) or as a civil servant, 8 people were self-employed, and 5 people worked at a privately-owned company. 59 mothers had a Bachelor’s Degree as their most recent education background, 12 people had an Associate’s Degree, 9 people had a High School Diploma, and 4 people had a Master’s Degree (Table 1).
The levels of vitamin D were classified into: deficiency (below 20 ng/mL), insufficiency (21-29 ng/mL), and normal (30-100 ng/mL). In this study, out of 84 pregnant mothers, 4 suffered from vitamin D deficiency (4.7%), 23 suffered from vitamin D insufficiency (26.2%), and 58 mothers had normal levels of vitamin D (69.1%). Out of all the pregnant mothers that suffered from either deficiency or insufficiency of vitamin D, 10 mothers were housewives, 7 people were employees at an SOE or worked as a civil servant, and 4 people were self-employed. Despite this, out of all of the pregnant mothers with normal vitamin D levels, the largest amount of occupation in this group were housewives, with 36 people (Table 2).
Figure 1: Study sample
Table 1: Data characteristics of ILTB pregnant women and non-ILTB pregnant women
Table 2: Classification of vitamin D levels of pregnant women based on occupation
The PCR electrophoresis results from the DNA isolate of the cytochrome gene CYP2R1 rs10741657 shows the DNA strand with the genotype variant GG (151bp, 105bp, 32bp), AA (256bp, 32bp), and GA (256 bp, 151bp, 105bp, 32bp) (Figure 2) and the PCR electrophoresis from the DNA isolate of the cytochrome gene CYP27B1 rs10877012 shows the DNA strand with the genotype variant GG (138bp, 49bp), TT (187bp), and GT (187bp, 138bp, 49bp) (Figure 3).
Figure 2: PCR-RFLP genotype CYP2R1
Figure 3: PCR-RFLP genotype CYP27B1
Table 3: Frequency distribution of gene polymorphisms in pregnant women
|
Genotype |
Pregnant Woman ILTB (n,%) |
Pregnant Woman non-ILTB (n,%) |
P-value |
|
CYP2R1 rs10741657 |
|
|
|
|
GG |
15 (35.7) |
16 (38.1) |
1.000 |
|
GA |
16 (38.1) |
17 (40.5) |
|
|
AA |
11 (21.4) |
9 (21.4) |
|
|
CYP27B1 rs10877012 |
|
|
|
|
GG |
9 (21.4) |
12 (28.6) |
0.604 |
|
GT |
28 (66.7) |
18 (42.9) |
|
|
TT |
5 (11.9) |
12 (28.1) |
|
Kolmogorov-Smirnov test.
Table 4: The average value of vitamin D levels in genotype variants in pregnant women
|
Varian genotype |
Frequency (n) |
Average of vitamin D(ng/mL) |
P-value |
|
CYP2R1 rs10741657 |
|
|
|
|
GG |
31 |
43.78 ± 29.17 |
0.871 |
|
GA |
33 |
44.34 ± 27.11 |
|
|
AA |
20 |
58.90 ± 59.19 |
|
|
CYP27B1rs10877012 |
|
|
|
|
GG |
21 |
46.61 ± 24.13 |
0.187 |
|
GT |
46 |
52.87 ± 47.31 |
|
|
TT |
17 |
34.55 ± 12.12 |
|
The polymorphism type for CYP2R1 rs10741657 and CYP27B1 rs10877012 were tested with Hardy-Weinberg Equilibrium (HWE) and a p-value of > 0.05 were obtained, which shows that this polymorphism is disequilibrium, so that the frequency of allele distribution in CYP2R1 and CYP27B1 were consistent with the HWE principle. On the CYP2R1 rs10741657, the frequency distribution of the GA heterozygous genotype variant was the largest variant found in LTBI pregnant mothers (38.1%) and non-LTBI pregnant mothers (40.5%). On the CYP27B1 rs10877012, the frequency distribution of the GT heterozygous genotype variant was the largest one found in LTBI pregnant mothers (66.7%) and non-LTBI pregnant mothers (42.9%) (Table 3).
On the polymorphism of CYP2R1 rs10741657, the highest mean value of vitamin D levels on pregnant mothers were found in the AA genotype, and the lowest mean value were found in the GA genotype, with a statistically insignificant difference (p = 0.871, p > 0.05). On the polymorphism of CYP27B1 rs10877012, the highest mean value of vitamin D levels on pregnant mothers were found in the GT genotype, and the lowest mean value were found in the TT genotype, with a statistically insignificant difference (p = 0.187, p > 0.05) (Table 4).
On the analysis of the correlation between genotype with vitamin D levels of pregnant mothers based on their LTBI status, the vitamin D levels on 42 pregnant mothers with LTBI-with the CYP2R1 rs10741657 polymorphism variance-the highest amount of genotype was the AA genotype, and the lowest genotype was the GG genotype (p = 0.903). Meanwhile, the mean value of vitamin D on 42 non-LTBI pregnant mothers, the highest amount of genotype was also the AA genotype, but the lowest amount of vitamin D levels on non-LTBI pregnant mothers were found on pregnant mothers with the GA genotype (p > 0.378, Table 5).
Table 5: Differences in mean vitamin D based on genotype in pregnant women
|
Genotype |
Average of vitamin D in Pregnant Woman ILTB ng/mL (frekuensi) |
P-value |
Average of vitamin D in Pregnant Woman non-ILTB ng/mL (frekuensi) |
P-value |
|
CYP2R1 rs10741657 |
|
|
|
|
|
GG |
40.55 ± 20.46 (15) |
0.903 |
46.80 ± 35.94 (16) |
0.378 |
|
GA |
43.64 ± 27.97 (16) |
|
44.99 ± 27.12 (17) |
|
|
AA |
63.35 ± 77.28 (11) |
|
53.47 ± 28.01 (9) |
|
|
CYP27B1 rs10877012 |
|
|
|
|
|
GG |
44.36 ± 19.21 (9) |
0.256 |
48.31 ± 27.98 (12) |
0.386 |
|
GT |
52.25 ± 52.82 (28) |
|
53.83 ± 38.62 (18) |
|
|
TT |
28.20 ± 6.40 (5) |
|
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