Evaluation of a new biomarker 14-3-3 Eta protein in diagnosis of rheumatoid arthritis
Lakshmi Deepika Yarlagadda1, Rachel Jacob1, D Liza Rajasekhar2, Krishna Mohan Iyyapu1, Sai Baba S S. Kompella1, Vijaya Bhaskar Madrol1, NN Sreedevi1, Siraj Ahmed Khan1, Noorjahan Mohammed1
1 Department of Biochemistry, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India
2 Department of Clinical Immunology and Rheumatology, Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India
|Date of Submission||17-Feb-2020|
|Date of Acceptance||07-May-2020|
|Date of Web Publication||3-Sep-2020|
Dr. Noorjahan Mohammed
Department of Biochemistry, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Telangana
Source of Support: None, Conflict of Interest: None
Context: The lack of sensitivity and specificity of the existing diagnostic markers in Rheumatoid arthritis (RA) stimulates the search for new biomarkers to improve diagnostic sensitivity especially in seronegative cases. Aims: To estimate the levels of 14-3-3 η in RA cases, to assess the positivity of 14-3-3 η in seronegative RA and to correlate rheumatoid factor (RF) positivity with 14-3-3 η positivity in seropositive RA. Settings and Design: This was cross sectional case control study by the Departments of Biochemistry and Rheumatology and clinical immunology. Subjects and Methods: A total of 61 RA cases and 20 healthy controls were included. Erythrocyte sedimentation rate, high sensitivity C reactive protein, RF, anti-citrullinated peptide antibodies (CCP) and 14-3-3 η protein were estimated. RA cases were further classified as seropositive (n = 23) and seronegative (n = 38) based on the positivity of either RF or anti-CCP result. Statistical Analysis: Prism 7 (GraphPad Software Inc.). Results: The median levels of 14-3-3 η (ng/ml) in cases (0.66 [0.2–1.1]) were significantly high compared to controls (0.1 [0.07–0.28]), P < 0.0001. It had a sensitivity of 74% and specificity of 90% at >0.3 ng/ml. 14-3-3 η showed statistically significant difference between seronegative cases and controls (P = 0.0003). The sensitivity and specificity of RF and anti-CCP were 33% and 85% and 37% and 90% respectively. The combination of 14-3-3 η, RF and anti-CCP showed sensitivity and specificity of 85.4% and 100% respectively with area under the curve 0.927. Conclusions: Our study demonstrated that serum levels of 14-3-3 η were significantly higher in cases compared to controls. Seronegative RA cases showed 14-3-3 η positivity in 71% of cases. 14-3-3 η appears to be a useful and highly specific marker in RA.
The diagnosis of RA is primarily based on clinical, radiological and immunological features.
The most frequent serological test is the measurement of rheumatoid factor (RF). Although the RF test has good sensitivity, it is not specific for RA, as it is often positive in healthy individuals and patients with other rheumatic or inflammatory diseases, autoimmune diseases or chronic infections.
Both RF and anti citrullinated peptide antibodies (CCP) antibodies (anti-cyclic citrullinated protein peptide antibodies) have been found to be present very early in the disease, often with the absence of clinical symptoms and many reports indicate that elevated levels of anti CCP antibodies can predict the development of erosive disease.,,,,, The sensitivity of the anti CCP test is around 50%–60% at the onset of RA and can rise as high as 85% later in the course of the disease. As 10%–15% patients with RA will be RF and anti-CCP antibody negative, there is a need for a new biomarker that will improve diagnostic sensitivity in these seronegative patients.
14-3-3 proteins represent a family of ubiquitously expressed intracellular chaperonins that are exclusively expressed in eukaryotic cells. The family consists of seven isoforms and share more than 50% amino acid homology between them: Beta (β), epsilon (ε), gamma (γ), eta (η), tau (τ), zeta (ζ), and sigma (σ). Serum 14-3-3 η is a novel joint-derived proinflammatory mediator implicated in the pathogenesis of RA. Unlike RF or anti CCP antibodies, which can arise as a consequence of disease in sites distinct from inflamed joints, 14-3-3 η hyper expression in RA is restricted to synovial joints with a significantly higher expression in synovial fluid than in serum. In established RA an association has been observed between the serum levels of 14-3-3 η and the degree of joint damage.
RA, like many other chronic diseases, has a significant impact on patient's functional ability and quality of life and it represents a huge economic burden, not only for patients and their families, but also for the society as a whole. The lack of sensitivity and specificity of the present diagnostic panel of parameters have led to the search for newer biomarkers to add to the present panel to identify the disease earlier and halt the progression of the disease and prevent the disability to greater extent.
So we aimed: (i) To estimate the levels of 14-3-3 η in established cases of RA and healthy controls, (ii) to assess the prevalence of 14-3-3 η in seronegative RA and also to correlate RF positivity with 14-3-3 η positivity in seropositive RA.
This study was cross sectional case control study conducted in the Department of Biochemistry in collaboration with Department of Clinical Immunology and Rheumatology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana. This study included 61 subjects aged between 18 and 60 years clinically diagnosed as RA as per American College of Rheumatology 2010 criteria as cases (taking into consideration joint involvement, serology, acute phase reactants (erythrocyte sedimentation rate [ESR], C-reactive protein [CRP]) and duration of symptoms) and 20 age and gender matched subjects as healthy controls. RA cases were further classified as seropositive (n = 23) and seronegative (n = 38) based on the positivity of either RF or anti-CCP result. Cut-off considered positive for RF and anti-CCP are >20 IU/ml and >5 U/ml respectively. Patients with bacterial or crystal-induced arthritis or a defined connective tissue disease or systemic vasculitis and patients not willing to provide informed consent were excluded.
After obtaining approval from Institutional Ethics Committee and taking informed written consent from the study subjects, 8 ml of blood was collected from each patient and healthy volunteers in plain and ethylenediaminetetraacetic acid tubes. Serum was separated after 1 h. ESR (Westergren method), high sensitivity CRP (hsCRP) (immunoturbidimetry), RF (IgM rhuematoid factor enzyme-linked immunosorbent assay [ELISA], Euroimmun, Germany), and Anti-CCP antibody (ELISA from HYCOR company) were estimated. Part of serum was aliquoted and stored at −80°C for subsequent analysis of 14-3-3 η protein by using Sandwich technique ELISA kit (Cusabio, Houston, Texas, USA).
A total of 81 subjects (61 RA cases and 20 healthy controls) were included in the study. Among 61 cases, 54 (89%) were females and 7 (11%) males. In the control group of 20 healthy subjects, there were 18 (90%) females and 2 (10%) males.
[Table 1] lists the demographic variables of controls and RA cases. Mean levels of ESR (mm/1st h) (48.6 ± 20.1 vs. 10.2 ± 5.4, P < 0.0001) were significantly higher in cases when compared to controls. Medians of hsCRP (mg/L) were significantly higher in RA cases when compared to controls (7.5 [3.5–21.6] and 2.3 [0.8–5.3], P = 0.0003) respectively [Figure 1].
Though median serum levels of 14-3-3 η were significantly higher in both seropositive and seronegative cases than in controls, there is no significant difference between these two groups (P > 0.99) [Figure 2].
Positivity of 14-3-3 η in seronegative and seropositive rheumatoid arthritis
Out of 61 RA patients included in our study, 38 were seronegative for RF and anti-CCP. Of these 38 patients, 27 were found to be positive for 14-3-3 η (with cut-off of >0.3 ng/ml) [Table 2]. Thus the prevalence of this marker in seronegative population in our study was found to be 71% whereas seropositive cases showed 78% positivity for 14-3-3 η.
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To assess the diagnostic performance of various biomarkers, ROC analysis was done. 14-3-3 η had a sensitivity of 74% and specificity of 90% at a cut-off of >0.3 ng/ml [Figure 3] for diagnosis of RA. RF had a sensitivity of 33% and specificity of 85% and AntiCCP has a sensitivity of 35% and specificity of 90% [Table 3].
|Figure 3: Comparison of receiver operator characteristics curves of various biomarkers|
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|Table 3: Utility of various markers in diagnosis of rheumatoid arthritis|
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The combination 14-3-3 η, RF and anti-CCP showed sensitivity and specificity of 85.42% and 100% respectively with AUC 0.927 [Figure 4].
|Figure 4: Receiver operator characteristics – Combination of biomarkers (14-3-3eta, rheumatoid factor and anti-citrullinated peptide antibodies)|
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As shown in [Table 4], serum 14-3-3 η has significant positive correlation with hsCRP. 14-3-3 η showed a positive correlation with RF (ρ = 0.42, P = 0.008) and anti-CCP (ρ = 0.51, P = 0.01) in seropositive RA cases.
|Table 4: Correlation of serum 14-3-3η with other variables in rheumatoid arthritis|
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Logistic regression analysis
When logistic regression is done, 14-3-3 η and ESR were found to be significant independent predictors of RA as shown in [Table 5].
Early diagnosis of RA has been a challenge for clinicians because symptoms are often subtle and can be similar to those of other diseases. Diagnosis and initiation of treatment of RA within 12 weeks of symptom onset, window of opportunity for therapy intervention, can only help prevent joint destruction and improve long-term function and patient prognosis. Unfortunately, many of these patients are not diagnosed during this time frame. The current laboratory markers used for diagnosis of RA are RF, anti-CCP, and CRP. These laboratory markers are normal in many patients, particularly in early disease. RF and anti-CCP have low sensitivity in early RA. In view of low sensitivity with current serological markers, there is need for better marker for early detection of RA. 14-3-3 protein is uniquely present in RA. This protein distributed throughout the body, particularly in synovium. Serum measurement of 14.3.3 η could play a valuable role in early diagnosis of RA to enable timely intervention.
This study has been undertaken to determine the effectiveness of the newer biomarker 14-3-3 η protein to diagnose more number of RA patients at an earlier stage. The synovial fluid determination of 14-3-3 η protein is more specific than the serum. As it is an invasive procedure, serum levels of 14-3-3 η protein have been assessed in this study which has been previously determined by many studies across the world.,
The median levels of 14-3-3 η in RA cases and healthy controls in our study are 0.66 ng/ml and 0.1 ng/ml, respectively [Figure 2]. The values are significantly higher in RA cases when compared to control group. 14-3-3 eta is a joint derived biomarker that is expressed at significantly higher levels in patients with RA than in healthy subjects, other autoimmune diseases, or viral and bacterial arthritides. In a study done by Maksymowych et al., the median 14-3-3 η levels are significantly higher in RA patients with established disease versus healthy subjects (1.12 vs. 0.001 ng/ml) and subjects with other arthropathies, connective tissue disorders and autoimmune diseases (0.02 ng/mL). In our study, ROC curve analysis of 14-3-3 η comparing RA cases to healthy subjects showed a sensitivity of 73.8% and specificity of 90% with AUC of 0.82. In a similar study done by Walter P. Maksymowych et al. sensitivity and specificity of 14-3-3 η was 77.0% and 92.6%, respectively. This difference in sensitivity could be due to low cut-off (≥0.19 ng/ml) as compared to our cut off (>0.3 ng/ml).
Comparison of 14-3-3 η, rheumatoid factor and anti-citrullinated peptide antibodies
Our study found a significant difference between established RA cases and healthy controls with respect to serum 14-3-3 η, RF and anti-CCP levels. 14-3-3 η has high sensitivity and specificity when compared to RF and anti-CCP. ROC-AUC for 14-3-3 η (0.82) is higher when compared to RF (0.59) and anti-CCP (0.68). In a study by Martinez-Prat et al., showed a sensitivity and specificity of 71.0% and 86.9%, respectively with AUC 0.82 (95% confidence interval [CI] 0.80–0.84) for anti-CCP. They also reported a sensitivity and specificity of 63.0% and 87%, respectively with AUC 0.79 (95% CI 0.77–0.81) for IgM RF. The high sensitivity for anti-CCP and RF in their study could be due to large sample size and low sensitivity in our study may be explained by inclusion of more number of seronegative cases. Out of 61 RA patients included in our study, 38 were seronegative for RF and anti-CCP. Of these 38 patients, 27 were found to be positive for 14-3-3 η making it a good marker in seronegative cases.
Utility of combination of markers
As anti-CCP and RF are often used together to diagnose RA, the benefit of adding 14-3-3 η to the existing markers was assessed. Addition of 14-3-3 η to RF and/or anti-CCP improved the sensitivity from 32.8% to 75% and from 35.4% to 83%, respectively. Combination all the three markers (RF + anti-CCP + 14-3-3 η) further increased the sensitivity to 85%. In the study conducted by W. P. Maksymowych et al. demonstrated that adding 14-3-3 η to anti-CCP resulted in an identification rate of 72% compared to 59% for anti-CCP alone. Adding RF to anti-CCP increased diagnostic sensitivity from 59% to 72% and this is-further increased to78% when 14-3-3 η was added.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]