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Year : 2018  |  Volume : 7  |  Issue : 3  |  Page : 242-246

Methylation status of alu repetitive elements in children with tuberculosis disease

Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Web Publication6-Sep-2018

Correspondence Address:
Dr. Mahadevan Subramanian
Department of Paediatrics, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_86_18

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Background: Investigation of DNA methylation in Alu repetitive elements (REs) was shown to be a promising field to explore transcriptional changes in human genome under disease condition. To scrutinize the association between Alu methylation and tuberculosis (TB) disease in children, the difference in Alu DNA methylation level was compared with healthy controls. Methods: Whole-blood genomic DNA from 36 TB-infected children and 32 healthy controls was isolated, and the level of Alu repeat DNA methylation was examined by methylation-specific polymerase chain reaction. Results: The median Alu methylation level in TB patients was 30% (Interquartile range [IQR], 25–30%), whereas in healthy controls, it was 75% (IQR, 50–75%) (P < 0.0001). The median level of DNA methylation of Alu RE in TB cases was significantly lower than healthy controls. Receiver operating characteristic curve analysis showed that the area under the curve for diagnosis was 0.969 (95% confidence interval, 0.936–1) (P < 0.0001), with 100% sensitivity and 84% specificity. Conclusion: Our results point out that detection of Alu DNA methylation in whole-blood DNA may be clinically useful tool for the diagnosis and prognosis of TB disease in children.

Keywords: Alu repeats, DNA methylation, hypomethylation, methylation-specific polymerase chain reaction, tuberculosis

How to cite this article:
Maruthai K, Subramanian M. Methylation status of alu repetitive elements in children with tuberculosis disease. Int J Mycobacteriol 2018;7:242-6

How to cite this URL:
Maruthai K, Subramanian M. Methylation status of alu repetitive elements in children with tuberculosis disease. Int J Mycobacteriol [serial online] 2018 [cited 2022 Jul 6];7:242-6. Available from: https://www.ijmyco.org/text.asp?2018/7/3/242/240700

  Introduction Top

One-third of the world population is infected with tuberculosis (TB) and approximately 10% people develop into active disease.[1] Around 2.8 million cases of TB were estimated in 2017 in India that makes quarter of the world's TB cases,[2] and TB is still a serious threat against children and it is estimated to be 10% of the global incidence and 250,000 death in 1.74 million in reported pediatric TB cases in 2016.[3]

The immune response against TB is a rather complex process and still emerging field of interest. The innate immune system exhibits initial immune defense against TB infection by pathogen recognition receptors [1],[4] that activate expression of several immune genes, cytokines, and interleukins.[5] Macrophages are the primary host cell target for tubercle bacilli for its intracellular growth and survival. Immune-primed macrophages are responsible for the activation of both innate and acquired immune responses [6] to eliminate tubercle bacilli before establishment of persistent infection.[5]

Several lines of regulatory mechanisms are underlying to regulate gene expression in human genome with respect to environmental cues. Transcription factors (TFs) are responsible for the controlled activation of genes under any circumstances such as pathophysiology of disease in particular.[7],[8] Transcriptional regulation of genes is crucial in cellular developmental process and it reshapes the transcription program of immune cells under infection particularly.[7]

Alu repeat elements are present up to 1.4 million copies in human genome [8] that comprises 11% of the total size.[8],[9] Alu repeats are 300-nucleotide long repeats which belong to SINE family and retrotranspositionally inactive.[9],[10],[11],[12] Due to its ample presence in human genome, these are normally found in gene-enriched regions and 3'UTR regions.[12] AluJ and AluS are the main subtypes in Alu family [11] that has several TF-binding sites, namely, MEF2 and ATF families and LXR and RAR nuclear receptors. These are the important receptor families which are consistent with macrophage response against stress and infection conditions.[8] Furthermore, Alu repeats contain certain common transcriptional motif that is recognized by various TFs such as SP1, p53, c-MYC, ANRIL, and NF-B.[8],[11]

Several reports suggest that DNA methylation in Alu repeats was associated with several diseases. The hypomethylation status of Alu repeats supports its retrotransposition from one place to another to disrupt active gene body and also lead to Alu-mediated recombination. It was reported that Alu retrotransposition contributes about 0.3% of all human diseases.[13]

In the present study, we used the methylation-specific polymerase chain reaction (MS-PCR) method to perceive the methylation status of Alu repeat elements in genomic DNA isolated from whole blood from TB patients compared to healthy controls.

  Methods Top

Ethics statement

The study protocol was approved by the Scientific Advisory Committee and Institute Ethical Committees. Blood samples were collected from TB-infected children and healthy donors after obtaining written informed consent.

Patients and specimen collection

Children with TB disease (<14 years, both genders) were recruited after obtaining informed consent. The inclusion criteria of both pulmonary TB (PTB) and extra-PTB (EPTB) patients included cartridge-based nucleic acid amplification test positive, smear positive, culture positive, chest X-ray positive, skin test positive, and clinical findings of TB disease. The inclusion criteria for healthy donors (<14 years, both genders) included no signs of TB and other infections, HIV negative, no surgical procedures underwent recently, asthma, and no other lung problems.

Genomic DNA isolation

Totally, 36 TB patients and 32 healthy controls were included in the study. Blood was collected from both groups and whole genomic DNA was isolated by FavorPrep Genomic DNA Mini Kit (Favorgen Biotech Corp., Taiwan) according to manufacturer's procedure. Briefly, 300 μl of whole blood was used to isolate genomic DNA according to the manufacturer's instructions. About 50 μl of elution buffer was used to elute DNA and stored at −20°C. NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to measure the concentration of genomic DNA.

Bisulfite conversion

Bisulfite conversion of genomic DNA was followed according to EZ DNA Methylation-Gold Kit (Zymo Research Inc., USA) manufacture's procedure. Briefly, 500 ng of genomic DNA from both cases and controls was used for bisulfite chemical conversion to differentiate unmethylated C to U and methylated C remains unchanged. Finally, 15 μl of elution buffer was used to elute bisulfite converted DNA and stored at −20°C.

Methylation standard DNA preparation

Fully methylated (M) and fully unmethylated (U) control DNA was purchased commercially (QIAGEN, Germany). M and U bisulfite-converted DNAs were mixed to obtain a panel of DNA standards, i.e. 100%, 75%, 50%, 25%, 10%, and 0% by combining the proportions of M and U human control DNA [Supplementary Table 1 [Additional file 1]].

CpG Island location and primer design

[Supplementary Figure 1 [Additional file 2]] shows the arrangement of consensus DNA sequence of Alu family and the location of CpG sites and CpG island (CGI) present in the given sequence. CGI in Alu repeats was identified by DBCAT tool (http://dbcat.cgm.ntu.edu.tw/), and MS-PCR primers were designed by MethPrimer tool with default settings.[14]

Methylation-specific polymerase chain reaction

Separate PCR reaction was performed specific for M and U MS-PCR analysis. A 20 μl of PCR mixture was prepared with 1 μl of bisulfite-converted DNA, 1 μl of each forward and reverse primers (10 pmol) from both methylated and unmethylated primers (separate reaction), 8 μl of ZymoTaq master mix, and 9 μl of nuclease-free water. PCR cycle reaction was performed with initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing [Supplementary Table 2 [Additional file 3]] for 35 s, and extension at 72°C for 30 s and a final extension at 72°C for 10 min.

Agarose gel electrophoresis

PCR product was separated in 1.5% agarose gel electrophoresis, and the intensity of the products and presence/absence of both M and U MS-PCR products were captured in ImageQuant LAS 500 (GE Healthcare, UK) and compared with control standards.

Statistical analysis

Categorical data were expressed as numbers and percentages. Percentage of Alu DNA methylation level was expressed in median with interquartile range (IQR) and Mann–Whitney t-test was performed to evaluate the difference of Alu DNA methylation level between cases and controls. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the cutoff value of % Alu DNA methylation in the study groups. All statistical analyzes were carried out in SPSS v19 and MS Excel at 95% confidence interval with P < 0.05 was considered as statistically significant.

  Results Top

Among the 36 cases, 18 (50%) were PTB and 18 (50%) were EPTB cases, and frequency of breakdown of EPTB cases enrolled in this study is represented in [Figure 1]. Baseline characteristics of the TB cases and healthy children enrolled in this study is represented in [Table 1].
Figure 1: Distribution of tuberculosis disease in study children (n = 36)

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Table 1: Baseline characteristics of the enrolled children

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Methylation of Alu elements in whole blood from tuberculosis patients and controls

Methylation-specific primers were successfully designed for Alu repeat elements, and methylation level of Alu repeats in whole-blood-derived DNA from TB-infected and healthy children was analyzed by MS-PCR. Methylation standard was prepared with mixture of both M and U DNA and subjected to MS-PCR procedure, and gel image was captured [Figure 2]A line a. This image was used to analyze and calculate the % Alu DNA methylation level in cases and controls.
Figure 2: Methylation-specific polymerase chain reaction results. (A) Comparison of whole-blood Alu DNA methylation levels between tuberculosis patients and controls. (B) Representative image methylation-specific polymerase chain reaction products. M indicates the fully methylated DNA used to prepare percentage proportion in methylation-specific polymerase chain reaction procedure. (a) Gel image of percentage of standard mDNA. (b) Methylation-specific polymerase chain reaction result of healthy controls. (c) Methylation-specific polymerase chain reaction result of tuberculosis cases. (d) Methylation-specific polymerase chain reaction result of tuberculosis cases – hypomethylation. (e) Unmethylation-specific methylation-specific polymerase chain reaction results of tuberculosis cases

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Significant difference in Alu DNA methylation level was observed between cases and healthy controls. The median of Alu methylation observed in cases was 30% (IQR, 25–30%) and in controls was 75% (IQR, 50–75%) (P < 0.0001) [Figure 2]B. This shows that control samples were hypermethylated Alu repeats than TB cases [Figure 2]A line b and [Figure 2]B. Occurrence of DNA methylation in Alu repeat sequences was observed both in cases and controls [Figure 2]A lines b and c. Hypermethylation was observed in healthy controls [Figure 2]A line b and hypomethylation was observed in TB cases [Figure 2]A line c.

Correspondingly, MS-PCR products for unmethylated PCR reaction were run paralleled and documented. Percentage of methylation and unmethylation was calculated by comparing the PCR products with methylation standards that run paralleled. Some of the cases and controls showed difference in Alu methylation level by MS-PCR. Some of the cases were observed only methylated (hypomethylated) bands in gel with complete absence of unmethylated bands [Figure 2]A lines d and e. No significant difference was seen in Alu DNA methylation level compared among cases with different diagnostic procedures [Table 2], whereas significant difference in Alu DNA methylation level was observed in cases with different diagnostic criteria when compared with healthy controls [Table 3].
Table 2: Comparison of percentage of Alu DNA methylation of tuberculosis diagnostic procedures versus controls

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Table 3: Comparison of percentage of Alu DNA methylation among tuberculosis diagnostic procedures

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Methylation of Alu elements in whole-blood pulmonary and extrapulmonary tuberculosis patients

To further determine the level of Alu DNA methylation difference between PTB and EPTB infection in children, MS-PCR analysis was done in all PTB and EPTB cases. PTB cases showed a median of Alu methylation level as 30% (IQR, 25–30%) and EPTB cases showed a median of Alu methylation level as 27.5% (IQR, 21.25%–30%). Significant difference in Alu methylation was observed in PTB cases when compared to controls [Table 2], whereas no significant difference in Alu methylation was observed between PTB and EPTB cases [Table 3]. Relatively, PTB cases were methylated than EPTB cases, but no significant difference was observed (P = 0.5) [Table 3]. Among EPTB cases, median of Alu methylation in TB meningitis cases (n = 10) was 27.5% (IQR, 10%–35%) versus others were 27.5% (IQR, 25–30%) (P = 0.9) [Table 3].

Diagnostic performance of DNA methylation level for tuberculosis disease

ROC curve was constructed to assess the feasibility of Alu DNA methylation for the diagnosis of TB patients [Figure 3]. We analyzed the methylation level of Alu in whole-blood-derived DNA between TB patients and healthy controls. The area under the curve (AUC) was 0.969 (95% confidence interval, 0.936–1) (P < 0.0001).
Figure 3: Receiver operating characteristic curve of Alu. The area under the curve was 0.969. Receiver operating characteristic showing cutoff value of percentage of Alu DNA methylation to distinguish between cases and controls (cutoff value = 40% with 100% sensitivity and 84% specificity, P < 0.0001)

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ROC results indicate that the methylation level of Alu in whole blood may have good diagnostic value with 100% sensitivity and 84% specificity at the optimal cutoff value of 40% in Alu DNA methylation.

  Discussion Top

Human genome exhibits hypomethylation in most CGIs in common to maintain its open chromatin state to influence nearby gene expression. On the contrary, hypermethylation was observed in repetitive elements (REs), such as LINEs, SINEs, and LTRs to avert its own transcription and transposition to maintain genome integrity.[15],[16] Human REs make up more than half of the human genome that covers approximately 50% of all CpG dinucleotides in the human genome.[16],[17] Thus, it is being a widely used method of averaging the methylation level of RE for global DNA methylation analysis.[16] Transcriptional regulation in eukaryotes maintains genome integrity and timely expression of genes required for developmental process. In mammals, the occurrence of methylation at the 5-C position of CpG dinucleotide in genomic DNA is an important epigenetic modification.[18] Impact of DNA methylation seen in gene expression, embryonic development, differentiation, genome imprinting, transposon silencing/activation, aging, and carcinogenesis is well documented.[17],[18]

The manifestation of DNA Methylation in Alu repeats from whole blood was analyzed both in cases and controls. No difference was observed between the disease condition and gender category. No significant difference in Alu methylation level was seen with clinical parameters included in this study [Table 3]. Both PTB and EPTB cases were hypomethylated than control samples [Table 2]. Four PTB and EPTB cases (hypomethylated) showed complete absence of unmethylation products, and three PTB and EPTB showed 10% Alu methylation products by MS-PCR.

Variation in the level of Alu DNA methylation was observed among cases (IQR, 25–30%); on the other hand, controls showed hypermethylation in Alu repeat elements (IQR, 50–75%). The variation among cases might be due to the disease condition, level of disease progression, genetic backup, type of disease, and organism specific. Hypermethylation in healthy controls indicates that the appearance of more methylation of cytosine residues in Alu DNA elements than TB cases. The occurrence of hypomethylation of Alu repeats in TB cases may play a substantial role in disease progression/containment in the host. The hypomethylation of Alu repeats in TB-infected patients widen the possibilities of appropriate expression of immune genes to control TB infection. On the contrary, it may influence the expression of immunosuppressive or anti-inflammatory genes to aid tubercle bacilli to forfend host immune response by unbalancing the immune response at post-TB infection. Hypomethylation of Alu elements was implicated in a variety of diseases, but its role in TB infection is not yet understood completely.

Recently, H3K4 monomethylation (H3K4 me1) of active enhancer regions were studied in THP-1 cells by ChIP assay and revealed that around 40% of the de novo regions analyzed which contained the presence of Alu repeat elements, especially enriched in AluJ and AluS subunits of Alu family. Interestingly, these de novo H3K4 me1 peaks were associated with genes mainly involving in host defense and apoptosis of infected cells primarily macrophages. This indicates the importance of Alu repeat elements in reshaping the transcriptional program of infected cells and early immune response of macrophages against TB infection in the host.[8] This result indicates that hypomethylation of Alu/Alu-associated elements is linked to TB disease prevention in infected cells, and our results also support the notion that hypomethylation of Alu elements may influence the expression of immune genes responsible for TB containment in host.

ROC was generated to scrutinize the diagnostic accuracy of Alu DNA methylation in whole blood between cases and healthy controls. A cutoff value of 40% of Alu DNA methylation was observed to differentiate TB cases from healthy controls with 100% sensitivity and 84% specificity. ROC curve confirmed that detection of Alu methylation by MS-PCR assay is a good diagnostic tool (AUC = 0.969) in TB infection in children and hence proved that Alu methylation quantification performed in this study has the ability to differentiate children with TB infection and healthy controls effectively.

  Conclusion Top

In the present study, hypomethylation of Alu repeat elements was observed in whole-blood genomic DNA derived from TB cases than healthy controls and demonstrated that detection of Alu methylation level may serve as a potential diagnostic and prognostic biomarker for TB infection in children. Although other gold standard TB diagnostic methods are still available with precise diagnostic potential, this Alu methylation detection also may have a good diagnostic value in future, and Alu hypomethylation might reflect the severity of TB disease in children. Further research should focus to identify and target specific CpG sites or CGI methylation by advanced technologies to improve the efficiency of the diagnostic method.

Financial support and sponsorship

The authors greatly acknowledge Jawaharlal Institute of Postgraduate Medical Education and Research Intramural Research grant for financial support.

Conflicts of interest

There are no conflicts of interest.

  References Top

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Blischak JD, Tailleux L, Mitrano A, Barreiro LB, Gilad Y. Mycobacterial infection induces a specific human innate immune response. Sci Rep 2015;5:16882.  Back to cited text no. 4
Cooper AM, Khader SA. The role of cytokines in the initiation, expansion, and control of cellular immunity to tuberculosis. Immunol Rev 2008;226:191-204.  Back to cited text no. 5
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]

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