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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 3  |  Issue : 3  |  Page : 238-241

Predictive Value of Beclin1 in the Pathogenesis of Rheumatoid Arthritis in the Indian Population


1 Department of Biochemistry, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India
2 Department of Medicine, All India Institute of Medical Sciences, Rishikesh, Uttarakhand, India

Date of Submission06-May-2022
Date of Decision08-Jun-2022
Date of Acceptance25-Jul-2022
Date of Web Publication28-Dec-2022

Correspondence Address:
Dr. Sarama Saha
Department of Biochemistry, All India Institute of Medical Sciences, Rishikesh - 249 203, Uttarakhand
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JME.JME_48_22

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  Abstract 


Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease leading to the destruction of articular cartilage and deformity of joints if not detected early. There is an unmet need to find out a highly sensitive and specific biomarker for RA. This was the first study designed in Indian setting to assess whether it can be used as a biomarker in the diagnosis of RA in the Indian population. Aim: To correlate serum Beclin1 in the pathogenesis of rheumatoid arthritis in the Indian population. Patients and Methods: Observational analytical study was conducted for 18 months at AIIMS Rishikesh, Department of Biochemistry in collaboration with the Department of Rheumatology. Beclin1 serum expression levels were estimated by the enzyme-linked immunosorbent assay and beclin1 mRNA expression was assessed by a real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMCs). Beclin1 expression was compared by Mann–Whitney U-test using SPSS 22 version. Cut-off values of Beclin1 for screening of cases were analysed by receiver operating characteristic test. Results: Age- and sex-matched 38 RA patients (5 males and 33 females) and 39 controls (8 males and 31 females) were recruited in the study. Patients with the American College of Rheumatology/European League Against Rheumatism score ≥6 were recruited in this study. Serum level of beclin1 was significantly (P ≤ 0.001) lower in cases (6.30 [2.82]) compared to healthy controls (11.43 [4.62]) which were corroborated with mRNA expression. The optimal cut-off value for detecting RA cases was 7.25 with 89.7% sensitivity and 79.8% specificity. Conclusion: Beclin1 may be involved in the pathogenesis of RA and may be considered a diagnostic marker for RA cases.

Keywords: Autophagy, Beclin1, DAS 28, receiver operating characteristic curve, rheumatoid arthritis


How to cite this article:
Varshney T, Singh PK, Saha S, Manna S, Pai VS, Naithani M, Mirza AA. Predictive Value of Beclin1 in the Pathogenesis of Rheumatoid Arthritis in the Indian Population. J Med Evid 2022;3:238-41

How to cite this URL:
Varshney T, Singh PK, Saha S, Manna S, Pai VS, Naithani M, Mirza AA. Predictive Value of Beclin1 in the Pathogenesis of Rheumatoid Arthritis in the Indian Population. J Med Evid [serial online] 2022 [cited 2023 Feb 1];3:238-41. Available from: http://www.journaljme.org/text.asp?2022/3/3/238/365881




  Introduction Top


Rheumatoid arthritis (RA) is a chronic, progressive, disabling systemic autoimmune disease which causes inflammation, swelling, pain in and around joints.[1] Signs and symptoms include tender inflamed joints associated with stiffness that is usually worse in the morning and also associated with inactivity, fatigue, fever and loss of appetite. RA can be difficult to diagnose in its early stage since signs and symptoms mimic other diseases. Untreated RA may lead to the development of complications such as osteoporosis, rheumatoid nodules, dry eyes and mouth, infections, carpal tunnel syndrome, heart problems (risk of hardened and blocked arteries), pleural disease and lymphoma.[2] The worldwide prevalence of RA is approximately 0.5%–1% among adults and increases to 5% in women over 70 years of age. RA is more common in women as compared to men. In India, the prevalence has been estimated to be 0.7%.[3] When RA progresses, it may lead to more disability and joint damage with the onset of comorbidities, decreased quality of life and premature mortality.[4]

Currently available biological markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and rheumatoid factor (RF) are not specific for the diagnosis of RA. Furthermore, anti–cyclic citrullinated peptide (CCP) is present only in 40%–60% of patients with RA.[5] Early detection and initiation of treatment can help patients to maintain their quality of life and prevent the severe progression of RA symptoms. Hence, there is an unmet need to find out one biomarker which would be more specific for the identification of disease activity of RA.

Autophagy is a physiological protective response to cell injury and helps in the restoration of cellular function through the degradation of dysfunctional cellular components. Beclin1 is considered to be the crucial component to initiate and regulate autophagy through activation of its downstream regulator such as microtubule-associated protein 1 light chain 3. A previous study revealed that Beclin1 siRNA-mediated inhibition of autophagy augmented cellular death through apoptosis.[6] Deregulated autophagy- and specific autophagy-related proteins have also been documented to contribute to the pathogenesis of chronic systematic autoimmune diseases such as RA.[7] Previous studies documented the possible connection between autophagy and osteoclastogenesis in mouse monocyte/macrophage cell lines.[8] Methotrexate commonly used for the treatment of RA patients protects synovial cells from apoptosis through induction of autophagy.[6] A recent study reported that autophagy hyperactivation may lead to methotrexate resistance which is an important mode of treatment for RA.[9] However, no study has been conducted to observe whether autophagy component, Beclin1 could be considered a biomarker for RA patients. Hence, this study has been carried out to observe the expression of beclin1 in the Indian populations.


  Materials and Methods Top


This was a clinic-based observational pilot study conducted in the Department of Biochemistry in collaboration with the rheumatology department at AIIMS, Rishikesh. Patients aged more than 18 years having well-established RA (based on the American College of Rheumatology/European League Against Rheumatism [ACR/EULAR] score) were included in the study. Patients having metabolic bone disease, malignancy or any other chronic diseases such as diabetes mellitus, thyroid disorder and receiving any disease-modifying drugs such as methotrexate were excluded from the study. Informed written consent was taken from every participant. After measurement of their anthropometric indices and assessment of disability index, 5 ml venous blood was drawn for the quantification of biochemical parameters such as ESR, CRP and expression (protein and mRNA) of Beclin1.

The patients having ACR/EULAR score ≥6 and who fulfilled inclusion/exclusion criteria were recruited for this study. Age- and sex-matched healthy volunteer participants, who were devoid of any chronic illness or recent acute illness history in the past 3 months, and no history of trauma, non-pregnant and non-lactating, were selected as controls.

ESR was estimated using the Westergren method, CRP by latex-enhanced immunoturbidimetric method and serum Beclin1 concentration was measured by (Enzyme-linked immunosorbent assay, G-Biosciences, USA). Total RNA was extracted from peripheral blood mononuclear cell (PBMC) using TRIzol® reagent (Invitrogen, Ambion by life technologies, USA). After the synthesis of complementary DNA (cDNA) using cDNA synthesis kit (4366596, applied biosciences USA) mRNA was quantified using quantitative reverse transcription polymerase chain reaction (PCR) with PCR conditions as follows: denaturation at 95°C for 2 h, annealing at 95°C for 15 s and extension at 60°C for 1 h repeated for 40 cycles. Primer sequences for Becin1 and housekeeping gene β-actin are shown in [Table 1].
Table 1: The primer sets of real-time reverse transcription-quantitative polymerase chain reaction

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Disease activity was assessed by DAS-28 score. DAS28 of >5.1 implies active disease, <3.2 indicate low disease activity and <2.6 is remission.

Statistical analysis

All the parameters were subjected to a normality test. After normality test parameters following Gaussian distribution were expressed as mean ± standard deviation. The parameter which did not follow the normal distribution was expressed as median (interquartile range). Comparison among parameters not following normal distribution was done by Mann–Whitney U-test and correlation among them was carried out by Spearman's correlation using SPSS 22 (SPSS version 22.0 for Windows, Armonk, NY: IBM Corp). Receiver operating characteristic (ROC) curve analysis was used to document the best cut-off value. Considering the confidence level as 95%, the statistical significance was defined as P < 0.05.


  Results Top


Thirty-eight RA patients including 5 (13%) males and 33 (87%) females and 38 healthy controls including 7 (19%) males and 31 (81%) females were recruited. The positive rate of serum RF in cases was found to be 76.92% and that of anti–CCP in cases was found to be 74.35%.

[Table 2] shows that cases were older than controls having a significant (P < 0.05) difference between cases (45.00 (24.25)) and controls (38.00 [7.00]). ESR showed a significant (P=<0.001) difference between cases [34.50 (28.25)] and controls [6.00 (6.00)]. DAS-28 ESR also showed a significant (P=<0.001) difference between cases [5.63 (2.49)] and controls [1.25 (0.55)].
Table 2: Comparison of various parameters measured in cases and healthy controls

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[Table 3] shows that Beclin1 was found to be significantly associated with ESR (r = 0.560, P ≤ 0.001) and DAS-28 ESR (r = 0.529, P ≤ 0.001). However, following age adjustment this significance disappeared with ESR (P = 0.216) and DAS-28 ESR (P = 0.170).
Table 3: Correlation of various parameters among total study participants (n=77) using Spearman's correlation

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Serum level of Beclin1 was significantly (P ≤ 0.001) lower in cases (6.30 [2.82]) compared to healthy controls (11.43 [4.62]) [Table 2] and it was corroborating with Beclin1 mRNA level [Figure 1].
Figure 1: Graphical representation of Beclin1 mRNA expression levels in healthy controls and RA patients. *P < 0.001. RA: Rheumatoid arthritis

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The ROC curve of the study participants is presented in [Figure 2]. The ROC curve showed that Beclin1 created a significantly very high area under the curve (AUC) 0.907 with 95% confidence interval, 0.835–0.979. The optimal cut-off point of Beclin1 for detecting RA cases corresponds to 7.25 with 89.7% sensitivity and 79.8% specificity.
Figure 2: ROC curve for the prediction of rheumatoid arthritis by Beclin 1. ROC: ROC: Receiver operating characteristic

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  Discussion Top


RA is a chronic progressive disabling disease, involving multiple systems. There is a sincere need to find a diagnostic marker with greater sensitivity and specificity so that the disease could be identified at an early stage.

A study conducted by Caramés et al. on osteoarthritis (OA) mice and human model documented the decreased expression of Beclin1 in OA chondrocytes and cartilage which was in agreement with our study.[10]

On the contrary, Lin et al. (2013) and Zhu et al. found increased levels of Beclin1 in osteoclasts and synovial tissue, respectively, in RA patients.[11],[12] The lower Beclin1 levels observed in our study could be attributed by the different sites of sample collection (serum vs. synovial tissue).

Intriguingly, we found correlated decreased levels of Beclin1 mRNA in RA patients as compared to controls. Hence, it suggests that regulation of Beclin1 level works at the transcription level as well.

Recent researches have provided a better understanding of environmental factors that influence RA risk, such as smoking and alcohol consumption. Other characteristics that have been linked to risk include birth weight, breastfeeding, socio-economic level and birth region. Because anti–citrullinated peptide antibodies status is linked to certain environmental elements, it is crucial to include Beclin1 in current and future research.[13] Hence, the discrepancy in our results may be due to different environmental factors and ethnicity in the Indian population.

As opposed to our finding, Kardideh B et al. (2019) observed higher expression of Beclin1 in serum. The discrepancy in study findings may be due to different laboratory setup, geographical variation and selection criteria for patients' recruitment. However, we consider our study findings are superior to that study because we have a larger number of participants (77 vs. 60) and we have documented similar expression both at protein and mRNA levels.[14]

Our study showed that Beclin1 level could not find any significant association with disease activity indicating that Beclin1 could not be used as a prognostic marker. However, considering the ROC curve analysis, it could be considered a diagnostic marker with high sensitivity and specificity. Since there was no study, where Beclin1 mRNA expression was documented from PBMC and predictive analysis was carried out to find out the cut-off value, comparison could not be done for verification of our study.

Zheng et al. reported that downregulation of Beclin1 expression was associated with poor prognosis in gastric carcinoma. They also suggested that Beclin1 could be considered a potential biomarker and be targeted for gene therapy in gastric cancer.[15] However, no study has documented the use of Beclin1 as a therapeutic target for RA cases.

The strength of the study to draw the maximum attention is that this was the first study to isolate Beclin1 from PBMC in RA patients of the Indian population and to conduct predictive analysis to find out the cut-off value where AUC showed the test of accuracy. However, there are some limitations. Since the risk factor for RA varies with geographical location and other environmental factors as well as our sample size was not large enough, this study's results could not be used to generalise in all Indian populations. Hence, a larger multicentric study is required. Moreover, the subjective assessment of symptoms by the participants may have created biasness in the categorisation of patients.


  Conclusion Top


The first study documented the corroborative lower expression of Beclin1 in the Indian population at protein as well as mRNA levels isolated from PBMC. It concludes that Beclin1 may be involved in pathogenesis of RA and could be a novel therapeutic target for treatment and improvement of quality of life in RA patients. Moreover, Beclin1 could be considered a diagnostic marker for the identification of RA cases having a cut-off value of 7.84 with 89.7% and 71.8% of sensitivity and specificity, respectively.

Acknowledgments

Tanu Varshney and Praveen Kumar Singh equally contributed to this article.

Financial support and sponsorship

AIIMS Rishikesh intramural fund was given to Dr. Sarama Saha.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Brazier Y. Rheumatoid arthritis (RA): Symptoms, Causes, and Complications. Medical News Today; 2018. Available from: https://www.medicalnewstoday.com/articles/323361.php. [Last accessed on 2022 Mar 10].  Back to cited text no. 1
    
2.
Mayo Clinic. Rheumatoid Arthritis. Mayo Foundation for Medical Education and Research; 2019. Available from: https://www.mayoclinic.org/diseases-conditions/rheumatoid-arthritis/symptoms-causes/syc-20353648. [Last accessed on 2022 Mar 10].  Back to cited text no. 2
    
3.
Akil M, Moots R. Rheumatoid arthritis: Clinical features and diagnosis. In: ABC of Rheumatology. 5th ed. Oxford: Wiley; 2018. p. 73-6.  Back to cited text no. 3
    
4.
Handa R, Rao UR, Lewis JF, Rambhad G, Shiff S, Ghia CJ. Literature review of rheumatoid arthritis in India. Int J Rheum Dis 2016;19:440-51.  Back to cited text no. 4
    
5.
Karkucak M, Çapkın E, Barçak ÖF, Çakırbay H, Tosun M, Gökmen F. Anti-CCP antibody and rheumatoid factor positivity in a case of tuberculosis arthritis. Arch Rheumatol 2010;25:95-8.  Back to cited text no. 5
    
6.
Vomero M, Barbati C, Colasanti T, Perricone C, Novelli L, Ceccarelli F, et al. Autophagy and rheumatoid arthritis: current knowledges and future perspectives. Front Immunol 2018;9:1577.  Back to cited text no. 6
    
7.
Shin YJ, Han SH, Kim DS, Lee GH, Yoo WH, Kang YM, et al. Autophagy induction and CHOP under-expression promotes survival of fibroblasts from rheumatoid arthritis patients under endoplasmic reticulum stress. Arthritis Res Ther 2010;12:R19.  Back to cited text no. 7
    
8.
Zhao Y, Chen G, Zhang W, Xu N, Zhu JY, Jia J, et al. Autophagy regulates hypoxia induced osteoclastogenesis through the HIF-1α/BNIP3 signaling pathway. J Cell Physiol 2012;227:639-48.  Back to cited text no. 8
    
9.
Xu K, Cai YS, Lu SM, Li XL, Liu L, Li Z, et al. Autophagy induction contributes to the resistance to methotrexate treatment in rheumatoid arthritis fibroblast-like synovial cells through high mobility group box chromosomal protein 1. Arthritis Res Ther 2015;17:374.  Back to cited text no. 9
    
10.
Caramés B, Taniguchi N, Otsuki S, Blanco FJ, Lotz M. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis. Arthritis Rheum 2010;62:791-801.  Back to cited text no. 10
    
11.
Lin NY, Beyer C, Gießl A, Kireva T, Scholtysek C, Uderhardt S, et al. Autophagy regulates TNFα-mediated joint destruction in experimental arthritis. Ann Rheum Dis 2013;72:761-8.  Back to cited text no. 11
    
12.
Zhu L, Wang H, Wu Y, He Z, Qin Y, Shen Q. The autophagy level is increased in the synovial tissues of patients with active rheumatoid arthritis and is correlated with disease severity. Mediators Inflamm 2017;2017:7623145.  Back to cited text no. 12
    
13.
Liao KP, Alfredsson L, Karlson EW. Environmental influences on risk for rheumatoid arthritis. Curr Opin Rheumatol 2009;21:279-83.  Back to cited text no. 13
    
14.
Kardideh B, Sadeghalvad M, Samimi Z, Mohammadi Motlagh HR, Taghadosi M. Evaluation of Beclin-1 and Atg5 genes expression levels in peripheral blood cells of patients with rheumatoid arthritis. KAUMS Journal (FEYZ).2019;23:135-42.  Back to cited text no. 14
    
15.
Zheng HC, Zhao S, Xue H, Zhao EH, Jiang HM, Hao CL. The roles of beclin 1 expression in gastric cancer: A marker for carcinogenesis, aggressive behaviors and favorable prognosis, and a target of gene therapy. Front Oncol 2020;10:613679.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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