|Ahead of print publication
Evaluation of the deleted in malignant brain tumor 1 protein expression and DNA methylation profile in rheumatoid arthritis patients
Khushboo Choudhury1, Monika Gandhi1, Uma Kumar2, Sayan Chatterjee1, Ram Singh Purty1
1 Cell and Molecular Biology Laboratory, University School of Biotechnology, Guru Gobind Singh Indraprastha University, New Delhi, India
2 Department of Rheumatology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||15-Aug-2021|
|Date of Acceptance||14-Nov-2021|
|Date of Web Publication||22-Jan-2022|
Ram Singh Purty,
University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C, Dwarka, New Delhi - 110 078
Source of Support: None, Conflict of Interest: None
Background: Rheumatoid arthritis (RA) is a chronic, autoimmune disease that progressively leads to joint destruction and functional disability which eventually affects the quality of life of the people suffering from it. Deleted in malignant brain tumor 1 (DMBT1) role has been explored in progression of Sjögren's syndrome (SS) and other autoimmune diseases, whereas it has yet to be reported in RA. In this study, we have analyzed and evaluated the expression of DMBT1 protein and the dmbt1 genes' methylation status in healthy volunteers, RA, and SS patients.
Methods: In the present investigation, the case–control study of 25 healthy volunteers and 17 RA- and 10 SS-confirmed patients was performed.
Results: It was observed that the concentration of DMBT1 protein in saliva decreases in both the disease conditions compared to healthy volunteers, while methylation status of dmbt1 gene was low in both patient cohorts in comparison to the healthy controls.
Conclusion : Results obtained from this study indicate that DMBT1 protein has the potential to qualify as a specific salivary biomarker as identified in RA and SS patients. DMBT1 protein can therefore be explored further as a noninvasive tool for diagnosis and prognosis.
Keywords: Deleted in malignant brain tumor 1, methylation, protein expression, rheumatoid arthritis, Sjögren's syndrome
|How to cite this URL:|
Choudhury K, Gandhi M, Kumar U, Chatterjee S, Purty RS. Evaluation of the deleted in malignant brain tumor 1 protein expression and DNA methylation profile in rheumatoid arthritis patients. Indian J Rheumatol [Epub ahead of print] [cited 2023 Feb 7]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=336271
| Introduction|| |
Rheumatoid arthritis (RA) is a chronic, systemic, long-term immune-mediated disease of unknown etiology that causes synovial inflammation and joint damage and ultimately leads to disability. In some individuals, the disease may also increase in severity leading to an involvement of major organs such as lungs, heart, eyes, and skin. RA belongs to the class of lifestyle diseases and affects up to 1% of the world's population. It is seen more frequently in females than in males, being predominantly observed in the older population.
Unlike RA, Sjögren's syndrome (SS) is far from a rare autoimmune exocrinopathy disorder, clinically distinguished by sicca symptoms developed by exocrine gland and extraglandular. The prevalence and characterization of RA associated with SS are poorly understood., SS and other rheumatic disorders may occur individually or in combination. There are many connective tissue disorders that SS overlaps with, RA being one of them. The incidence of overlap ranges from 3.8% to 38.7% in various parts of the world. Therefore, in the present study, we have taken SS as a positive control. An overlap complication is also observed in SS propounding the relation between the RA and SS pathophysiologies which could also point to the development of SS later on in serious RA patients.
Altered sialochemistry in SS patients carries a risk of oral diseases by altering the antimicrobial properties of proteins, electrolytes, small molecules, and salivary immunoglobulins. One class of proteins that promotes bacterial agglutination and clearance, is the salivary scavenger and agglutinin (SALSA) protein also known as deleted in malignant brain tumor 1 (DMBT1) or gp340. Its role as a pattern recognition molecule has been recognized in the innate immune system. DMBT1 has been noted to agglutinate cariogenic bacteria like Streptococcus mutans.
DMBT1 exists in most mucosal surfaces such as mouth, lungs, and vagina and is released in saliva and tears. It is a member of the scavenger receptor cysteine-rich group B protein superfamily,, and its gene is present on the chromosome 10q25.3–26.1 and is found within one of the putative intervals for tumor suppressor genes. The DMBT1 protein is involved in mucosal innate immunity through binding to various pathogens and host molecules, including surfactant protein, immunoglobulin A, and complement component 1q. SALSA was ultimately speculated to have an influence in epithelial homeostasis, innate immunity, and inflammation. The dmbt1 gene shows regular genomic modifications and has been assumed to play a significant function in the autoimmune diseases, because of which it is coherent and conceivable to consider a connection of DMBT1 to RA.
Several studies have shown the altered methylation status of dmbt1 gene in cancer and other autoimmune diseases such as Sjögren's syndrome, allergic rhinitis, ulcerative colitis, and Crohn's disease., We analyzed the dmbt1 gene methylation status in RA and SS aiming to provide new insights in its epigenetic regulation. Identification and quantification of dmbt1 methylation levels in RA can serve as a potential area for research and could also be a probable target for the development of noninvasive RA diagnosis.
| Methods|| |
Study design and subject information
In the present study, 25 healthy volunteers and 17 seropositive RA (rheumatoid factor and anti-citrullinated protein antibodies) and 10 SS-confirmed patients were enrolled at All India Institute of Medical Sciences (AIIMS), New Delhi. Clinical records of all patients were reviewed in detail, and informed consent was obtained for each volunteer for the study (healthy and diseased). Institutional Ethics Committee (IEC) approval from GGSIPU (Ref no. GGSIPU/IEC/2017/) and IEC approval from AIIMS (Ref no. IEC-630/November 3, 2017, RP-39/2017) were obtained for this current study. The inclusion and exclusion criteria are included in [Table 1].
Saliva samples were collected from the healthy volunteers and patients. 2.5 ml of saliva was collected 2 h after the intake of food, into sample collection tubes (Cat. No.:523080, Tarsons, India) to minimize the dilution of protein of interest and prevent any food debris mixing with saliva sample. To minimize the salivary protein degradation, we placed the tube on ice and Protease Cocktail Inhibitor (Cat. No.: P8340, Merck, Darmstadt, Germany) (1 μl/ml of the whole saliva) was added immediately. The supernatant was collected and stored at −80°C.
Ethylenediaminetetraacetic acid-coated tubes/vacutainers (Cat. No.: 368856, BD Biosciences, USA) were used to collect the blood. Total 3 ml of blood was taken and each blood sample was centrifuged at 2000 rpm for 10 min to separate the plasma, and the separated plasma was stored at −80°C.
Quantifying deleted in malignant brain tumor 1 protein in saliva and plasma
For the quantification of DMBT1 protein, saliva and plasma samples were used. The collected saliva and plasma were diluted with a dilution buffer in a ratio of 1:10 and 1:2, respectively. Standard reactions were performed as per the manufacturer protocol (FineTest Cat. No. EH1756 [Hubei, China]). The detection range was 0.313–20 ng/ml. DMBT1 level was detected using 100 μl of samples, and all reactions were set in triplicate in the respective well and incubated at 37°C for 90 min. After incubation, well contents were discarded and the plate was washed twice with a washing buffer provided with the kit. Biotin-labeled antibody (100 μl) was added to the well and incubated at 37°C for 60 min, followed by three washes. HRP-Streptavidin Conjugate (SABC; 100 μl) was added to the wells and incubated at 37°C for 30 min, followed by five washes. TMB substrate (90 μl) was added next to each well and incubated for 10–15 min in the dark at 37°C. Finally, 50-μl stop solution was added to each well, and absorbance was taken at 450 nm in a microplate reader immediately.
DNA methylation profiling
For DNA methylation profiling, DNA from blood was extracted using the QiAamp Blood Mini DNA extraction kit (Cat No./ID: 51104, QIAGEN, USA) and quantified by NanoDrop (Thermo Fisher, USA). Genomic DNA was converted to bisulfite DNA using EZ DNA Methylation™ kit according to manufacturer's protocol (Zymo Research, USA; Cat. No. D5001). 200 ng of genomic DNA was used for bisulfite treatment and purified in 20 μl of M-Elution Buffer. The bisulfite DNA was later used as a template in polymerase chain reaction (PCR). For amplification of dmbt1 gene promoter region, the following primers were used: 5'-TGAATTTTTTAAGATTAAAATTAAGTT-3' as forward primer and 3'-AAAAAATAAAAACTTTTACTAACAATC-5' as reverse primer. Both the primers were designed using MethPrimer tool. Touchdown PCR was carried out with the following conditions – first cycle of initial denaturation at 94°C for 5 min followed by annealing at 56°C for 30 s and extension at 72°C for 30 s, three cycles' of annealing at 55°C keeping extension temperature at 72°C for 30 s, next three cycles' annealing at 54°C, again three cycles' annealing at 52°C with extension temperature and time remaining constant, 51°C annealing for three cycles, and finally, annealing temperature at 50°C for 25 cycles with extension at 72°C for 5 min. The PCR product obtained was sequenced bidirectionally. The chromatogram of the sequenced data was visualized using the Chromas software (Technelysium Pty. Ltd., Australia; version 2.6.6). Using the software, BioEdit (BioEdit 7.2), multiple sequence alignment was carried out and the methylation percentage was calculated using the formula: methylation percentage = (peak height of C/peak height of C + T) ×100.
SPSS 20 program (IBM Corp., Armonk, NY, USA) and STATA 15.1 program were used. Two-sample t-test with equal variances and Wilcoxon rank-sum test were employed for statistical analysis. Sensitivity versus specificity for area under curve–receiver operating characteristic (AUC-ROC) curve analysis had been done for comparative analysis.
| Results|| |
Demographic characteristics of the patients
All the patients and volunteers recruited for this study were from North India, specifically Delhi-NCR. The healthy cohort consisted of 7 males and 18 females with age 35 years ± 10.11. Of the 17 patients recruited with RA, 2 were males and 15 were females with age 41.8 years ± 10.33, and all SS patients recruited were females, with age 41.5 years ± 10.89.
Deleted in malignant brain tumor 1 protein in saliva and plasma
There was a significant difference in saliva DMBT1 protein levels between RA and SS patient groups when compared to the healthy controls; the concentration of salivary DMBT1 protein was lowered in both diseases. In healthy controls, the DMBT1 protein level in saliva is 1600 μg/ml, whereas for RA and SS, the levels were 174 μg/ml and 198 μg/ml, respectively [Figure 1]. It was observed that DMBT1 protein in saliva of SS patients presents an AUC value of 0.8632 on the AUC-ROC curve [Figure 2]a. Similarly, applying the same test on the salivary DMBT1 protein levels of RA patients, the AUC value obtained is 0.8148. Using these values, it can be seen that the DMBT1 protein levels in saliva of RA patients provide a strong positive discrimination against healthy population [Figure 2]b.
|Figure 1: Deleted in malignant brain tumor 1 protein level depiction in saliva of healthy controls, rheumatoid arthritis patients, and Sjögren's syndrome patients. The total protein content in healthy controls was 1600 μg/ml; in rheumatoid arthritis patients, the protein content was 174 μg/ml; and in Sjögren's syndrome patients, the total protein amount was 198 μg/ml|
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|Figure 2: (a) Area under curve–receiver operator characteristic curve for deleted in malignant brain tumor 1 protein in saliva of rheumatoid arthritis patients. The area under curve value for this test was 0.8148, which indicates an effectiveness of this criteria as a preliminary marker. (b) Area under curve–receiver operator characteristic curve for deleted in malignant brain tumor 1 protein in saliva of Sjögren's syndrome patients. The indicated value of area under curve was 0.8632 in Sjögren's syndrome patients meaning it has better corroboration of the use of deleted in malignant brain tumor 1 protein as an initial, crude marker|
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In the case of plasma, DMBT1 protein level was found to be significantly increased in both RA and SS conditions compared to healthy population [Figure 3]. When the protein was quantified in plasma, it was interesting to find that in healthy samples, the DMBT1 protein was found to be lowest (1.99 μg/ml), elevated in RA patients (5.04 μg/ml), and highest in SS patients (7.35 μg/ml).
|Figure 3: Deleted in malignant brain tumor 1 protein level in plasma of healthy controls, rheumatoid arthritis patients, and Sjögren's syndrome patients. The total protein content in healthy controls was 1.99 μg/ml; in rheumatoid arthritis patients, the protein content was 5.04 μg/ml; and in Sjögren's syndrome patients, the total protein amount was 7.35 μg/ml|
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Methylation status of deleted in malignant brain tumor 1 gene in rheumatoid arthritis and Sjögren's syndrome patients
A PCR product of 209 bp was obtained after bisulfite PCR and separated on 2% agarose gel. After bisulfite sequencing, it was noted that there was a significant decrease in the methylation level in both diseased conditions, against the healthy individuals [Figure 4]. The dmbt1 gene was 71.7% methylated in healthy controls, while in RA and SS patients, the methylation percentage was 55.45% and 60.31%, respectively.
|Figure 4: The percentage of methylation of the deleted in malignant brain tumor 1 gene across all three sample cohorts. An average of site-specific methylation percentage was calculated across all three cohorts. As seen from the graph, the highest methylation of the gene is seen in healthy population while the methylation is lowered in both diseases|
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To establish the diagnostic value of the dmbt1 gene methylation in patients' cohorts, the AUC-ROC analysis was employed. The AUC value of dmbt1 gene was 0.85 and 0.78 for RA and SS patients, respectively [Figure 5]a and [Figure 5]b. The result obtained corroborated the correctness of diagnosis, and these findings can act as preliminary indicators for having the potential to diagnose the disease.
|Figure 5: (a) Area under curve–receiver operator characteristic curve of methylation of deleted in malignant brain tumor 1 gene for the rheumatoid arthritis patients. (b) Area under curve–receiver operator characteristic curve of methylation of deleted in malignant brain tumor 1 gene of Sjögren's syndrome patients. The area under curve value of deleted in malignant brain tumor 1 was 0.85 and 0.78 for rheumatoid arthritis and Sjögren's syndrome patients, respectively|
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| Discussion|| |
DMBT1 protein has been shown to play a crucial role as a tumor suppressor gene in cancers such as gastric cancer, cervical cancer, gallbladder cancer, colorectal cancer, and acute lymphoblastic leukemia; it plays a protective role in allergic rhinitis and is also involved in pathogenic clearance from the oral mucosa. Studies have demonstrated that there are a number of molecular and immunological markers in saliva, that have unlocked various arenas for research into salivary proteomics. Moreover, previous studies suggested that these salivary constituents may actually be effective indicators of both local and systemic diseases.
In this study, the DMBT1 protein level in saliva was observed to be highest in healthy volunteers while it was significantly downregulated in both patient cohorts. It has been described in SS that lack of salivation plays a role in development of infection. It may be reasonable to postulate that reduction in the levels of DMBT1 in saliva in diseased states may lead to an impairment of the innate immune system regulation too. Due to possible uptake of DMBT1 by the lectin pathway, there could be a plausible reduction in its levels in saliva. When the levels of DMBT1 in plasma were quantified, it was interesting to note that in both RA and SS, DMBT1 protein levels were increased in both disease cohorts compared to the healthy levels. This could point to the fact that in diseased states, there exists a dysregulation of the complement cascade pathways due to which DMBT1 bioavailability was elevated.
There exists a transcriptional silencing of promoter elements in hypermethylation, observed in studies. It has been observed that there exists hypomethylation in RA fibroblast like synoviocytes (FLS) cells. For establishment of the diagnostic value of the dmbt1 gene methylation in both patient cohorts, we used the AUC-ROC analysis to observe if methylation signature could be used as a marker for disease. The AUC value of dmbt1 was 0.85 and 0.78 for RA and SS patients, respectively. The findings from this study could help us to further understand the underlying factors that could lead to full-blown RA. Upon validation of these results on a greater set of patients, the results could possibly lead us toward the establishment of DMBT1 as a marker for RA and other diseases.
Our findings would further require validation through independent studies on tissues with dmbt1 gene expression and with a greater number of patients. There might exist some conditions where factors that are not under observation could also affect the consequences of epigenetics. Addressing the fundamental question of epigenetic epidemiology, it would be interesting to delve deeper into the mechanisms linking genetics to epigenetics and further to phenotype.
Diagnosis and monitoring of RA often require painful invasive procedures such as repeated blood withdrawal and synovial aspiration, thereby adding undue stress to the patient. Using a salivary marker could ease the painful experience associated with RA to correctly arrive at a diagnosis. It is difficult to establish the diagnosis of RA and SS since there is no single diagnostic gold standard test available. Those available could show signs of overlap too. For research purposes, several classification methods have been described, and the most widely used are the 2016 revised ACR and the EULAR criteria. Rheumatic disease diagnosis is mostly carried out by excluding a wide range of other diseases, without pathognomonic tests and/or criteria. Hence, even after fulfillment of classification criteria, the diagnosis could be revised upon emergence of new symptoms or newer treatment options. Our proposed molecular marker may serve the purpose in this case to the extent of confirming RA and SS patients and help in early start of treatment, but further validation is needed to correctly elucidate its use as an indicator for RA or SS.
| Conclusion|| |
The study of methylation status and the quantification of the DMBT1 protein and gene has helped us build a preliminary foundation for further understanding the factors that affect RA. DMBT1 protein levels are reduced in diseased states such as RA and SS, while it is much higher in healthy population. However, DMBT1 is observed to be elevated in plasma of the same patients and reduced in healthy controls. Considering that the study of the methylation status of the dmbt1 gene was preliminary, it can help us lay the foundation for more in-depth work. A rapid, multiplex, and miniaturized analytical assay could be established using salivary DMBT1. This would greatly reduce the dependency of diagnostic tests on invasive procedures.
The authors acknowledge the funding agency (FRGS-GGSIPU) for financial support. KC also acknowledges the STRF-GGSIPU fellowship.
This study was approved by the IEC of Guru Gobind Singh Indraprastha University, New Delhi, and AIIMS, New Delhi, India.
Financial support and sponsorship
The study is funded by FRG Scheme received from GGS Indraprastha University, New Delhi, India (Grant No. GGSIPU/DRC/Ph.D/Adm./2017/516; GGSIPU/DRC/FRGS/2018/20 (115); GGSIPU/DRC/FRGS/2019/22).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]