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Diagnostic value and clinical significance of anti-carbamylated protein (anti-CarP) antibodies in Egyptian patients with rheumatoid arthritis
The Egyptian Rheumatologist xxx (xxxx) xxx
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The Egyptian Rheumatologist journal homepage: www.elsevier.com/locate/ejr
Diagnostic value and clinical significance of anti-carbamylated protein (anti-CarP) antibodies in Egyptian patients with rheumatoid arthritis Samar R. Mohamed a, Nanis O. Neseem a,⇑, Shereen S. Metwally b, Basma A. El-Kady a a b
Rheumatology and Rehabilitation Department, Faculty of Medicine, Mansoura University, Egypt Clinical Pathology and Immunology Department, Faculty of Medicine, Mansoura University, Egypt
a r t i c l e
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Article history: Received 27 March 2019 Accepted 19 April 2019 Available online xxxx Keywords: Anti-carbamylated protein (Anti-CarP) antibodies Egyptian Rheumatoid arthritis DAS28 HAQ Modified Larsen score
a b s t r a c t Aim of the work: The study was performed to assess the diagnostic potential of anti-carbamylated proteins (anti-CarP) antibodies in Egyptian patients with RA and their relation to clinical manifestations, laboratory findings and radiological damage. Patients and methods: The study involved 90 RA patients and 90 matched healthy controls. Disease activity score (DAS28) and health assessment questionnaire (HAQ) were assessed. Plain x-ray hands and feet were performed and assessed by modified Larsen’s score. Laboratory investigations including acute phase reactants, rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) and anti-CarP antibodies were estimated. Results: The patients mean age was 42.6 ± 10.4 years (20–60 years), disease duration was 4.3 ± 3.3 years and were 82 females (91.1%) and 8 males (8.9%). The mean DAS28 was 4.9 ± 1.2, HAQ was 1.6 ± 0.7 and Larsen score 33.8 ± 12.2. 29/90 (32.2%) patients were positive for anti-CarP antibodies and were significantly associated with extra-articular manifestations and deformity (p < 0.001), but not with acute phase reactants. Anti-CarP antibodies significantly associated with modified Larsen’s (p < 0.001), but not DAS28 (p = 0.13). The sensitivity and specificity were 32.2% and 96.7% for anti-CarP antibodies, 61.1% and 97.8% for anti-CCP antibodies, 66.7% and 91.1% for RF respectively. 25.7% of anti-CCP negative patients showed anti-CarP positivity, and radiological damage was significantly associated with anti-CarP in them. Conclusion: The sensitivity of anti-CarP antibodies was low in comparison to both RF and anti-CCP; however, they showed high specificity and were detected in anti-CCP negative patients, so they could be useful to test beside anti-CCP and RF. Anti-CarP antibodies may reflect disease radiological damage. Ó 2019 Publishing services provided by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
1. Introduction Rheumatoid arthritis (RA) is a common autoimmune disease manifested by inflammation of the joints that leads to damage of bone and cartilage. However, the exact pathogenesis is not fully clear till now [1]. Several gene polymorphisms have been implicated in the development of the disease in Egyptian patients [2–4]. Early diagnosis of RA is a central issue in clinical practice, as it leads to better disease control, reduction of permanent disability which is important long term consequence of the disease, and stopping radiological progression [5]. Many Egyptian studies on RA investigated the role of different biomarkers in determining the disease activity and clinical significance [6–10], yet few have focused on the diagnostic potential [11]. Peer review under responsibility of Egyptian Society of Rheumatic Diseases. ⇑ Corresponding author. E-mail address: [email protected] (N.O. Neseem).
The human leucocytic antigen (HLA-DRB1 04) was found to be related with RA in Egyptian patients and was strongly linked to the enhanced levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies that are related to the disease development, severity and activity [12]. Anti-CCP was found to have a role in detecting erosive arthritis in Egyptian systemic lupus erythematosus (SLE) patients [13]. Beside anti-CCP, other autoantibodies are also found in RA patients and target structurally similar determinants [5]. Anti-mutated citrullinated vimentin (anti-MCV) was significantly associated with the functional disability and radiologic progression in Egyptian RA patients; and was considered a potential biomarker [14]. Even though it was remarkably increased in hepatitis C virus (HCV) patients, anti-MCV had no role in the detection of HCV-associated arthritis [15]. Antibodies that recognize carbamylated proteins are called anti-carbamylated protein (anti-CarP) antibodies [5]. Citrullination and carbamylation are post-translational modifications which end in the production of citrulline and homocitrulline respectively [16].
https://doi.org/10.1016/j.ejr.2019.04.004 1110-1164/Ó 2019 Publishing services provided by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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S.R. Mohamed et al. / The Egyptian Rheumatologist xxx (xxxx) xxx
In both, a neutrally charged amino acid replaces a positively charged one. They differ structurally in length; homocitrulline is one CH3 group longer [17]. Anti-CarP antibodies were found in RA patients [18]. Interestingly they are detected in patients negative for anti-CCP [17]. Additionally, they were related to developing RA in arthralgia patients [19]. Similar to anti-CCP, anti-CarP antibodies were found in serum prior to appearance of clinical manifestations of RA [20]. In a recent study on Egyptian psoriatic arthritis patients, it was suggested that anti-CarP antibody may represent a promising biomarker to predict joint damage and disease activity [21]. Several questions are still open regarding antiCarP antibodies and whether they could further elucidate RA pathogenesis and predict clinical disease [22]. The study was carried out to assess the diagnostic potential of anti-carbamylated proteins (anti-CarP) antibodies in Egyptian patients with RA and study their relation to clinical manifestations, laboratory findings and radiological damage.
Table 1 Sociodemographic characteristics, clinical, and laboratory findings and scores of the RA patients.
2. Patients and methods
RA: rheumatoid arthritis, VAS: visual analogue scale, DAS28: disease activity score, HAQ: health assessment questionnaire, ESR: erythrocyte sedimentation rate, CRP: C-reactive protein, RF: rheumatoid factor, antiCCP: anti-cyclic citrullinated peptide, anti-CarP: anticarbamylated protein.
The study included ninety RA patients who were recruited from the Physical Medicine, Rheumatology and Rehabilitation outpatient clinic at Mansoura University Hospital. The range of the patients’ age was 20–60 years and they were 82 females (91.1%) and 8 males (8.9%). The patients fulfilled the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for RA [23]. Patients excluded were those having other autoimmune diseases, kidney disease, cancers, viral hepatitis, and other erosive joint diseases as gout, erosive osteoarthritis and seronegative spondyloarthritis. 90 healthy subjects of matching age and sex were considered as a control group. A written informed consent was obtained from all participants and was approved by the Mansoura research ethics committee. All patients were subjected to detailed history taking, full clinical examination, assessment of joint pain by visual analogue scale (VAS), of disease activity score (DAS28) [24] and of the health assessment questionnaire (HAQ) [25]. Plain x-ray of hands and feet were done for all patients to assess radiological damage by modified Larsen’s score [26]. Laboratory investigations were done and included the complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-CCP. Anti-carbamylated protein antibody were detected in serum by Human anti-carbamylated protein Antibody (ACPAb) ELISA kit (Sunred Biological Technology Ca., Ltd, Shanghai China). Statistical analysis: SPSS for windows version 20.0 (SPSS, Chicago, IL) was used to perform the statistical analyses. Before performing any calculations, data were tested for normality of distribution. Data were expressed as mean ± standard deviation (SD) or number and percentage. The comparisons were determined using Student’s t test or Mann-Whitney U test for variables with continuous data of normal and abnormal distribution respectively. Chi-square test was used for comparing variables with categorical data. The diagnostic accuracy of the three autoantibodies was identified. Receiver operating characteristic (ROC) curves were performed, and area under the curve (AUC) is calculated to measure of the usefulness of the three autoantibodies in identification of cases with RA. P < 0.05 was considered significant. 3. Results The mean age of the 90 RA patients was 42.6 ± 10.4 years (20–60 years) and were 82 females (91.1%) and 8 males (8.9%). The 90 control were matched for age (41.9 ± 9.8 years) and gender 81 female (90%) and 9 male (10%). The sociodemographic characteristics of the patients are presented in Table 1. The presence of
Variable
RA patients (n = 90)
Age (years) Females:Males Disease duration (years) Morning stiffness (min.) Tender joint count Swollen joint count VAS-pain (mm) DAS28 HAQ ESR (mm) CRP (mg/dl) RF (IU/ml) positivity Anti-CCP (unit/ml) Positivity Anti-CarP positivity Modified Larsen score
42.6 ± 10.4 82/8 (91.1/8.9) 4.3 ± 3.3 54.1 ± 46.2 6.4 ± 4.1 3.3 ± 2.7 45.3 ± 19.4 4.9 ± 1.2 1.6 ± 0.7 44.5 ± 21.3 25.5 ± 24.7 52.8 ± 61.8 60 (66.7) 47.3 ± 37.7 55 (61.1) 29 (32.2) 33.8 ± 12.2
anti-CarP, RF, and anti-CCP in patients and control is shown in Table 2. Distribution of patients according to the existence of RF, anti-CCP and anti-CarP in their sera is shown in Table 3. AntiCarP was positive in 9 (25.7%) patients of the 35 with a negative anti-CCP. There is no gender differences (p = 0.65). Anti-CarP positive RA patients showed significant association with extra-articular manifestations, and deformity (p < 0.001). On the other hand, anti-CarP positivity was insignificantly associated with disease duration, duration of morning stiffness (MS), tender joint count (TJC), swollen joint count (SJC), VAS, ESR, or CRP (p ˃ 0.05) (Table 4). The association of anti-CarP antibody with the DAS28, HAQ and modified Larsen are presented in Table 5. The modified Larsen’s score was significantly higher in patients with positive anti-CarP and negative for anti-CCP (n = 9) (43.8 ± 9.9) compared to 61 cases with a negative anti-CarP (28.7 ± 10.3) (P < 0.001). The potential value of anti-CarP as a diagnostic marker for RA compared to RF and anti-CCP antibodies are presented in Table 6. The highest test for accuracy was that of anti-CCP antibody (79.4%). The ROC curves showed that anti-CCP had highest AUC than both anti-CarP and RF. The AUC was 0.77 for anti-CCP, and 0.68 for both anti-CarP and RF. 4. Discussion Recognition of autoantibodies specific of RA offered a great help in better disease understanding and in categorization of patients [18]. Anti-CarP antibodies were found to expect RA occurrence in arthralgia patients. They also could be detected in the patient’s sera long time preceding the clinical manifestations of the disease [19]. In the current study, anti-CarP antibodies were detected in almost a third of the RA patients. This is consistent with the findings of another study which reported that 45% and 43% of RA patients were positive for anti-CarP IgG and IgA respectively [18]. In other studies anti-CarP was found positive in 30% [27], 34.4% [28], 29.4% [29], 44.9% [30], and 41.9% [31] of RA patients. In this study, 7.8% were positive for anti-CarP only and 5.6% for anti-CCP. In addition, anti-CarP antibodies were found in 25.7% of anti-CCP negative RA patients. This supports the findings in other studies in which anti-CarP antibodies were detected in anti-CCP negative patients [18,28,29,32,33]. This indicates that
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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S.R. Mohamed et al. / The Egyptian Rheumatologist xxx (xxxx) xxx Table 2 Comparison of presence of autoantibodies in rheumatoid arthritis patients and control. +ve autoantibody n (%)
RA (n = 90)
RF Anti-CCP Anti-CarP
60 55 29
Control (n = 90) (66.7) (61.1) (32.2)
8 2 3
Chi square test
(8.9) (2.2) (3.3)
v2
p
63.9 72.1 25.7
<0.001 <0.001 <0.001
RA: rheumatoid arthritis, RF: Rheumatoid factor, Anti-CCP: anti cyclic citrullinated peptide, Anti-CarP: anti-carbamylated protein. Bold values are significant at p < 0.05.
Table 3 Distribution of the rheumatoid arthritis patients according to the presence of antibodies in their sera. Subset n (%)
RA patients (n = 90)
Seronegative RF + ve only Anti-CCP + ve only Anti-CarP + ve only RF and anti-CCP + ve RF and anti-CarP + ve anti-CCP and anti-CarP + ve RF, anti-CCP and anti-CarP + ve
(17) (9) (5) (7) (30) (2) (1) (19)
18.9 10.0 5.6 7.8 33.3 2.2 1.1 21.1
RA: rheumatoid arthritis, RF: Rheumatoid factor, Anti-CCP: anti cyclic citrullinated peptide, Anti-CarP: anti-carbamylated protein. Bold values are significant at p < 0.05.
Table 4 Association of anti-carbamylated protein with clinical and laboratory parameters in rheumatoid arthritis patients. Parameter
Anti-CarP in RA patients (n = 90)
mean ± SD or n(%)
negative (n = 61)
positive (n = 29)
p
Disease duration (years) Morning stiffness (min.) Tender joint count Swollen joint count Visual analogue scale (mm) Extrarticular manifestations Deformities ESR (mm/1st hr) C-reactive protein (mg/dl)
8.3 ± 2.9 101.7 ± 46.1 9.8 ± 3.6 7.0 ± 2.5 43.6 ± 19.9 18 (29.5) 18 (29.5) 40.4 ± 20.5 27.0 ± 10.1
8.4 ± 3.9 114.9 ± 41.9 11.2 ± 4.8 8.0 ± 3.0 49.0 ± 18.2 21 (72.4) 19 (65.5) 53.1 ± 20.8 30.2 ± 14.8
0.81 0.2 0.13 0.1 0.22 <0.001 <0.001 0.56 0.23
Anti-CarP: anti-carbamylated protein, ESR: erythrocyte sedimentation rate. Bold values are significant at p < 0.05.
Table 5 Association of anti-CarP antibody with the disease activity score, health assessment questionnaire and the modified Larsen score in rheumatoid arthritis patients. Score mean ± SD
Anti-CarP in RA patients (n = 90)
DAS28 HAQ Modified Larsen
4.9 ± 1.2 1.48 ± 0.7 28.7 ± 10.3
+ve (n = 29)
ve (n = 61) 5.3 ± 1.1 1.94 ± 0.5 44.3 ± 9.0
p 0.13 0.003 <0.001
Anti-CarP: anti-carbamylated protein, DAS28: disease activity score, HAQ: Health assessment questionnaire, Bold value are significant at p < 0.05.
anti-citrullinated and anti-homocitrullinated proteins antibodies are mostly non cross reactive as previously reported [34]. The chief benefit of detection of multiple disease biomarkers either simultaneously or alternatively is obtaining a more thorough classification of RA subtypes which increases the accuracy of diagnosing RA [35]. The anti-CarP antibodies had low sensitivity (32.2%) of diagnostic potential compared to RF and anti-CCP. As regard the specificity, anti-CCP was the highest and was comparable to that of anti-CarP antibodies. Moreover, anti-CCP had highest AUC than both antiCarP and RF. In a recent meta-analysis it has been reported that anti-CarP sensitivity and specificity were 42% and 96% respectively in comparison to control [36], while in an Iraqi study anti-CarP sensitivity and specificity for diagnosis of RA were 46% and 97.1% respectively [37]. In another study, anti-CarP sensitivity and specificity were 44% and 89% respectively with an AUC of 0.67 [38] which was close to the present result. Similarly, in an Italian RA cohort, anti-CarP sensitivity was 46.8% and specificity 91.95% while the AUC was highest for the anti-CCP [28] as in this work. Our study found no significant association between anti-CarP antibodies and disease duration, duration of MS, TJC, SJC, VAS, ESR or CRP. Conversely, anti-CarP were significantly associated with extra-articular manifestations and deformity. In concordance, a study performed to assess the diagnostic value of anti-CarP antibodies in Iraqi patients reported absence of significant relation between anti-CarP and the duration of disease SJC, TJC, and ESR [37]. A significant relationship between anti-CarP and deformity was reported by others [39]. The anti-CarP antibodies did not associated with the disease activity in the current patients which support the findings of others [27,31,39]. In contrast, it was demonstrated that higher disease activity levels were significantly associated with anti-CarP in cases of inflammatory polyarthritis. Also, in their work, anti-CCP but not RF showed significant association with DAS28 [40]. However, that study was conducted on a large number of patients and assessed the associations between anti-CarP antibody status and longterm disease activity. This study suggested a relation of anti-CarP antibodies with poor prognosis as evidenced by the significant association between them and both radiological damage and functional disability. The relation of anti-CarP with erosions and radiological damage was also documented in other several studies [1,18,27,29,39]. More interestingly, the association of anti-CarP with radiological damage was found to be significant in anti-CCP negative patients, which may support the suggestion of the clinical usefulness of anti-CarP antibodies as a prognostic marker in anti-CCP negative patients
Table 6 Diagnostic value of different autoantibodies in rheumatoid arthritis patients. Antibody
Anti-CarP Anti-CCP RF
RA patients (n = 90) Sensitivity (95% CI)
Specificity (95% CI)
PPV
NPV
Acc.
32.2% (22.8–42.9) 61.1% (50.3–71.2) 66.7% (56.0–76.3)
96.7% (90.6–99.3) 97.8% (92.2–99.7) 91.1% (83.2–96.1)
90.6% 96.5% 82.2%
58.8% 72.5% 73.2%
64.4% 79.4% 78.9%
RA: rheumatoid arthritis, Anti-CarP: anti-carbamylated protein, Anti-CCP: anti cyclic citrullinated peptide, RF: rheumatoid factor, CI: confidence interval, PPV: positive predictive value, NPV: negative predictive value, ACC: accuracy.
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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and their relation to the risk of developing a more severe disease in this subgroup of RA patients [40]. In conclusion, the study confirmed the presence of anti-CarP antibodies in RA patients, and inspite of having low sensitivity; they were detected in patients negative for anti-CCP and thus could have an additive diagnostic value beside RF and anti-CCP. Anti-CarP antibodies may serve as good predictors of disease severity and are related to joint damage even in anti-CCP negative patients. More studies are required to reveal the diagnostic utility of anti-CarP antibodies especially during early stages of RA. Conflict of interest None. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Brink M, Verheul MK, Rönnelid J, Berglin E, Holmdahl R, Toes RE, et al. Anticarbamylated protein antibodies in the pre-symptomatic phase of rheumatoid arthritis, their relationship with multiple anti-citrulline peptide antibodies and association with radiological damage. Arthritis Res Ther 2015;17(1):25. [2] Hegazy MK, Auf FA, Neseem NO, Al-Harrass MF. Toll-like receptor (TLR9) -1486 T/C (rs187084) gene polymorphism in Egyptian patients with rheumatoid arthritis. The Egyptian Rheumatologist. 2018;epub ahead of print. [3] Shaker OG, El-Demellawy HH, Salem MN, Eesa NN. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity. Egyptian Rheumatologist 2016;38(4):283–8. [4] Ayeldeen G, Nassar Y, Ahmed H, Shaker O, Gheita T. Possible use of miRNAs146a and -499 expression and their polymorphisms as diagnostic markers for rheumatoid arthritis. Mol Cell Biochem 2018 Dec;449(1–2):145–56. [5] Trouw LA, Huizinga TW, Toes RE. Autoimmunity in rheumatoid arthritis: different antigens-common principles. Ann Rheum Dis 2013;72:ii132-6. [6] Allam SI, Sallam RA, Elghannam DM, El-Ghaweet AI. Clinical significance of serum B cell chemokine (CXCL13) in early rheumatoid arthritis patients. The Egyptian Rheumatologist 2019;41(1):11–4. [7] Hameed AK, El-Said TO, Askar HY, ElKady BA. Performance of serum adenosine deaminase in measuring disease activity in rheumatoid arthritis patients. The Egyptian Rheumatologist. 2018;epub ahead of print. [8] El Defrawy AO, Gheita TA, Raslan HM, El Ansary MM, El Awar AH. Serum and synovial cartilage oligomeric matrix protein levels in early and established rheumatoid arthritis. Z Rheumatol 2016;75(9):917–23. [9] Gheita TA, Sayed S, Gheita HA, Kenawy SA. Vitamin D status in rheumatoid arthritis patients: relation to clinical manifestations, disease activity, quality of life and fibromyalgia syndrome. Int J Rheum Dis 2016;19(3):294–9. [10] Gheita TA, Kenawy SA, El Sisi RW, Gheita HA, Khalil H. Subclinical reduced G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients: relation to clinical characteristics, disease activity and metabolic syndrome. Mod Rheumatol 2014;24(4):612–7. [11] Hussein N, Kamel N, Fouda N, Farrag D. Peptidyl-arginine deiminase-type IV as a diagnostic and prognostic marker in rheumatoid arthritis patients. The Egyptian Rheumatologist. 2018; epub ahead of print. [12] Soliman AF, Egaila SE, Ali AI, Azab NI, Al-Gohary HH. HLA-DRB1 alleles in Egyptian rheumatoid arthritis patients: Relations to anti-cyclic citrullinated peptide antibodies, disease activity and severity. Egyptian Rheumatologist 2016;38(4):269–75. [13] Abdel-Magied RA, AbuOmar HA, Ali LH, Talaat H, Mohamed FI. Diagnostic potential of ultrasound in systemic lupus erythematosus patients with joint involvement: Relation to anticyclic citrullinated peptide (anti-CCP), disease activity and functional status. The Egyptian Rheumatologist. 2018;epub ahead of print. [14] El-Shazly RI, Hussein SA, Raslan HZ, Elgogary AA. Anti-mutated citrullinated vimentin antibodies in rheumatoid arthritis patients: Relation to disease activity and manifestations. Egyptian Rheumatologist 2014;36(2):65–70. [15] El-Fedawy S, Ghareeb H, Lotfy N, El-Azizi NO, Mahmoud A. Role of antimutated citrullinated vimentin antibodies in chronic hepatitis C patients and its relation to HCV associated arthritis. Egyptian Rheumatologist 2017;39 (3):175–8. [16] Pruijn GJ. Citrullination and carbamylation in the pathophysiology of rheumatoid arthritis. Front Immunol 2015;6:192.
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Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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Diagnostic value and clinical significance of anti-carbamylated protein (anti-CarP) antibodies in Egyptian patients with rheumatoid arthritis Samar R. Mohamed a, Nanis O. Neseem a,⇑, Shereen S. Metwally b, Basma A. El-Kady a a b
Rheumatology and Rehabilitation Department, Faculty of Medicine, Mansoura University, Egypt Clinical Pathology and Immunology Department, Faculty of Medicine, Mansoura University, Egypt
a r t i c l e
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Article history: Received 27 March 2019 Accepted 19 April 2019 Available online xxxx Keywords: Anti-carbamylated protein (Anti-CarP) antibodies Egyptian Rheumatoid arthritis DAS28 HAQ Modified Larsen score
a b s t r a c t Aim of the work: The study was performed to assess the diagnostic potential of anti-carbamylated proteins (anti-CarP) antibodies in Egyptian patients with RA and their relation to clinical manifestations, laboratory findings and radiological damage. Patients and methods: The study involved 90 RA patients and 90 matched healthy controls. Disease activity score (DAS28) and health assessment questionnaire (HAQ) were assessed. Plain x-ray hands and feet were performed and assessed by modified Larsen’s score. Laboratory investigations including acute phase reactants, rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) and anti-CarP antibodies were estimated. Results: The patients mean age was 42.6 ± 10.4 years (20–60 years), disease duration was 4.3 ± 3.3 years and were 82 females (91.1%) and 8 males (8.9%). The mean DAS28 was 4.9 ± 1.2, HAQ was 1.6 ± 0.7 and Larsen score 33.8 ± 12.2. 29/90 (32.2%) patients were positive for anti-CarP antibodies and were significantly associated with extra-articular manifestations and deformity (p < 0.001), but not with acute phase reactants. Anti-CarP antibodies significantly associated with modified Larsen’s (p < 0.001), but not DAS28 (p = 0.13). The sensitivity and specificity were 32.2% and 96.7% for anti-CarP antibodies, 61.1% and 97.8% for anti-CCP antibodies, 66.7% and 91.1% for RF respectively. 25.7% of anti-CCP negative patients showed anti-CarP positivity, and radiological damage was significantly associated with anti-CarP in them. Conclusion: The sensitivity of anti-CarP antibodies was low in comparison to both RF and anti-CCP; however, they showed high specificity and were detected in anti-CCP negative patients, so they could be useful to test beside anti-CCP and RF. Anti-CarP antibodies may reflect disease radiological damage. Ó 2019 Publishing services provided by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
1. Introduction Rheumatoid arthritis (RA) is a common autoimmune disease manifested by inflammation of the joints that leads to damage of bone and cartilage. However, the exact pathogenesis is not fully clear till now [1]. Several gene polymorphisms have been implicated in the development of the disease in Egyptian patients [2–4]. Early diagnosis of RA is a central issue in clinical practice, as it leads to better disease control, reduction of permanent disability which is important long term consequence of the disease, and stopping radiological progression [5]. Many Egyptian studies on RA investigated the role of different biomarkers in determining the disease activity and clinical significance [6–10], yet few have focused on the diagnostic potential [11]. Peer review under responsibility of Egyptian Society of Rheumatic Diseases. ⇑ Corresponding author. E-mail address: [email protected] (N.O. Neseem).
The human leucocytic antigen (HLA-DRB1 04) was found to be related with RA in Egyptian patients and was strongly linked to the enhanced levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies that are related to the disease development, severity and activity [12]. Anti-CCP was found to have a role in detecting erosive arthritis in Egyptian systemic lupus erythematosus (SLE) patients [13]. Beside anti-CCP, other autoantibodies are also found in RA patients and target structurally similar determinants [5]. Anti-mutated citrullinated vimentin (anti-MCV) was significantly associated with the functional disability and radiologic progression in Egyptian RA patients; and was considered a potential biomarker [14]. Even though it was remarkably increased in hepatitis C virus (HCV) patients, anti-MCV had no role in the detection of HCV-associated arthritis [15]. Antibodies that recognize carbamylated proteins are called anti-carbamylated protein (anti-CarP) antibodies [5]. Citrullination and carbamylation are post-translational modifications which end in the production of citrulline and homocitrulline respectively [16].
https://doi.org/10.1016/j.ejr.2019.04.004 1110-1164/Ó 2019 Publishing services provided by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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S.R. Mohamed et al. / The Egyptian Rheumatologist xxx (xxxx) xxx
In both, a neutrally charged amino acid replaces a positively charged one. They differ structurally in length; homocitrulline is one CH3 group longer [17]. Anti-CarP antibodies were found in RA patients [18]. Interestingly they are detected in patients negative for anti-CCP [17]. Additionally, they were related to developing RA in arthralgia patients [19]. Similar to anti-CCP, anti-CarP antibodies were found in serum prior to appearance of clinical manifestations of RA [20]. In a recent study on Egyptian psoriatic arthritis patients, it was suggested that anti-CarP antibody may represent a promising biomarker to predict joint damage and disease activity [21]. Several questions are still open regarding antiCarP antibodies and whether they could further elucidate RA pathogenesis and predict clinical disease [22]. The study was carried out to assess the diagnostic potential of anti-carbamylated proteins (anti-CarP) antibodies in Egyptian patients with RA and study their relation to clinical manifestations, laboratory findings and radiological damage.
Table 1 Sociodemographic characteristics, clinical, and laboratory findings and scores of the RA patients.
2. Patients and methods
RA: rheumatoid arthritis, VAS: visual analogue scale, DAS28: disease activity score, HAQ: health assessment questionnaire, ESR: erythrocyte sedimentation rate, CRP: C-reactive protein, RF: rheumatoid factor, antiCCP: anti-cyclic citrullinated peptide, anti-CarP: anticarbamylated protein.
The study included ninety RA patients who were recruited from the Physical Medicine, Rheumatology and Rehabilitation outpatient clinic at Mansoura University Hospital. The range of the patients’ age was 20–60 years and they were 82 females (91.1%) and 8 males (8.9%). The patients fulfilled the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria for RA [23]. Patients excluded were those having other autoimmune diseases, kidney disease, cancers, viral hepatitis, and other erosive joint diseases as gout, erosive osteoarthritis and seronegative spondyloarthritis. 90 healthy subjects of matching age and sex were considered as a control group. A written informed consent was obtained from all participants and was approved by the Mansoura research ethics committee. All patients were subjected to detailed history taking, full clinical examination, assessment of joint pain by visual analogue scale (VAS), of disease activity score (DAS28) [24] and of the health assessment questionnaire (HAQ) [25]. Plain x-ray of hands and feet were done for all patients to assess radiological damage by modified Larsen’s score [26]. Laboratory investigations were done and included the complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-CCP. Anti-carbamylated protein antibody were detected in serum by Human anti-carbamylated protein Antibody (ACPAb) ELISA kit (Sunred Biological Technology Ca., Ltd, Shanghai China). Statistical analysis: SPSS for windows version 20.0 (SPSS, Chicago, IL) was used to perform the statistical analyses. Before performing any calculations, data were tested for normality of distribution. Data were expressed as mean ± standard deviation (SD) or number and percentage. The comparisons were determined using Student’s t test or Mann-Whitney U test for variables with continuous data of normal and abnormal distribution respectively. Chi-square test was used for comparing variables with categorical data. The diagnostic accuracy of the three autoantibodies was identified. Receiver operating characteristic (ROC) curves were performed, and area under the curve (AUC) is calculated to measure of the usefulness of the three autoantibodies in identification of cases with RA. P < 0.05 was considered significant. 3. Results The mean age of the 90 RA patients was 42.6 ± 10.4 years (20–60 years) and were 82 females (91.1%) and 8 males (8.9%). The 90 control were matched for age (41.9 ± 9.8 years) and gender 81 female (90%) and 9 male (10%). The sociodemographic characteristics of the patients are presented in Table 1. The presence of
Variable
RA patients (n = 90)
Age (years) Females:Males Disease duration (years) Morning stiffness (min.) Tender joint count Swollen joint count VAS-pain (mm) DAS28 HAQ ESR (mm) CRP (mg/dl) RF (IU/ml) positivity Anti-CCP (unit/ml) Positivity Anti-CarP positivity Modified Larsen score
42.6 ± 10.4 82/8 (91.1/8.9) 4.3 ± 3.3 54.1 ± 46.2 6.4 ± 4.1 3.3 ± 2.7 45.3 ± 19.4 4.9 ± 1.2 1.6 ± 0.7 44.5 ± 21.3 25.5 ± 24.7 52.8 ± 61.8 60 (66.7) 47.3 ± 37.7 55 (61.1) 29 (32.2) 33.8 ± 12.2
anti-CarP, RF, and anti-CCP in patients and control is shown in Table 2. Distribution of patients according to the existence of RF, anti-CCP and anti-CarP in their sera is shown in Table 3. AntiCarP was positive in 9 (25.7%) patients of the 35 with a negative anti-CCP. There is no gender differences (p = 0.65). Anti-CarP positive RA patients showed significant association with extra-articular manifestations, and deformity (p < 0.001). On the other hand, anti-CarP positivity was insignificantly associated with disease duration, duration of morning stiffness (MS), tender joint count (TJC), swollen joint count (SJC), VAS, ESR, or CRP (p ˃ 0.05) (Table 4). The association of anti-CarP antibody with the DAS28, HAQ and modified Larsen are presented in Table 5. The modified Larsen’s score was significantly higher in patients with positive anti-CarP and negative for anti-CCP (n = 9) (43.8 ± 9.9) compared to 61 cases with a negative anti-CarP (28.7 ± 10.3) (P < 0.001). The potential value of anti-CarP as a diagnostic marker for RA compared to RF and anti-CCP antibodies are presented in Table 6. The highest test for accuracy was that of anti-CCP antibody (79.4%). The ROC curves showed that anti-CCP had highest AUC than both anti-CarP and RF. The AUC was 0.77 for anti-CCP, and 0.68 for both anti-CarP and RF. 4. Discussion Recognition of autoantibodies specific of RA offered a great help in better disease understanding and in categorization of patients [18]. Anti-CarP antibodies were found to expect RA occurrence in arthralgia patients. They also could be detected in the patient’s sera long time preceding the clinical manifestations of the disease [19]. In the current study, anti-CarP antibodies were detected in almost a third of the RA patients. This is consistent with the findings of another study which reported that 45% and 43% of RA patients were positive for anti-CarP IgG and IgA respectively [18]. In other studies anti-CarP was found positive in 30% [27], 34.4% [28], 29.4% [29], 44.9% [30], and 41.9% [31] of RA patients. In this study, 7.8% were positive for anti-CarP only and 5.6% for anti-CCP. In addition, anti-CarP antibodies were found in 25.7% of anti-CCP negative RA patients. This supports the findings in other studies in which anti-CarP antibodies were detected in anti-CCP negative patients [18,28,29,32,33]. This indicates that
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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S.R. Mohamed et al. / The Egyptian Rheumatologist xxx (xxxx) xxx Table 2 Comparison of presence of autoantibodies in rheumatoid arthritis patients and control. +ve autoantibody n (%)
RA (n = 90)
RF Anti-CCP Anti-CarP
60 55 29
Control (n = 90) (66.7) (61.1) (32.2)
8 2 3
Chi square test
(8.9) (2.2) (3.3)
v2
p
63.9 72.1 25.7
<0.001 <0.001 <0.001
RA: rheumatoid arthritis, RF: Rheumatoid factor, Anti-CCP: anti cyclic citrullinated peptide, Anti-CarP: anti-carbamylated protein. Bold values are significant at p < 0.05.
Table 3 Distribution of the rheumatoid arthritis patients according to the presence of antibodies in their sera. Subset n (%)
RA patients (n = 90)
Seronegative RF + ve only Anti-CCP + ve only Anti-CarP + ve only RF and anti-CCP + ve RF and anti-CarP + ve anti-CCP and anti-CarP + ve RF, anti-CCP and anti-CarP + ve
(17) (9) (5) (7) (30) (2) (1) (19)
18.9 10.0 5.6 7.8 33.3 2.2 1.1 21.1
RA: rheumatoid arthritis, RF: Rheumatoid factor, Anti-CCP: anti cyclic citrullinated peptide, Anti-CarP: anti-carbamylated protein. Bold values are significant at p < 0.05.
Table 4 Association of anti-carbamylated protein with clinical and laboratory parameters in rheumatoid arthritis patients. Parameter
Anti-CarP in RA patients (n = 90)
mean ± SD or n(%)
negative (n = 61)
positive (n = 29)
p
Disease duration (years) Morning stiffness (min.) Tender joint count Swollen joint count Visual analogue scale (mm) Extrarticular manifestations Deformities ESR (mm/1st hr) C-reactive protein (mg/dl)
8.3 ± 2.9 101.7 ± 46.1 9.8 ± 3.6 7.0 ± 2.5 43.6 ± 19.9 18 (29.5) 18 (29.5) 40.4 ± 20.5 27.0 ± 10.1
8.4 ± 3.9 114.9 ± 41.9 11.2 ± 4.8 8.0 ± 3.0 49.0 ± 18.2 21 (72.4) 19 (65.5) 53.1 ± 20.8 30.2 ± 14.8
0.81 0.2 0.13 0.1 0.22 <0.001 <0.001 0.56 0.23
Anti-CarP: anti-carbamylated protein, ESR: erythrocyte sedimentation rate. Bold values are significant at p < 0.05.
Table 5 Association of anti-CarP antibody with the disease activity score, health assessment questionnaire and the modified Larsen score in rheumatoid arthritis patients. Score mean ± SD
Anti-CarP in RA patients (n = 90)
DAS28 HAQ Modified Larsen
4.9 ± 1.2 1.48 ± 0.7 28.7 ± 10.3
+ve (n = 29)
ve (n = 61) 5.3 ± 1.1 1.94 ± 0.5 44.3 ± 9.0
p 0.13 0.003 <0.001
Anti-CarP: anti-carbamylated protein, DAS28: disease activity score, HAQ: Health assessment questionnaire, Bold value are significant at p < 0.05.
anti-citrullinated and anti-homocitrullinated proteins antibodies are mostly non cross reactive as previously reported [34]. The chief benefit of detection of multiple disease biomarkers either simultaneously or alternatively is obtaining a more thorough classification of RA subtypes which increases the accuracy of diagnosing RA [35]. The anti-CarP antibodies had low sensitivity (32.2%) of diagnostic potential compared to RF and anti-CCP. As regard the specificity, anti-CCP was the highest and was comparable to that of anti-CarP antibodies. Moreover, anti-CCP had highest AUC than both antiCarP and RF. In a recent meta-analysis it has been reported that anti-CarP sensitivity and specificity were 42% and 96% respectively in comparison to control [36], while in an Iraqi study anti-CarP sensitivity and specificity for diagnosis of RA were 46% and 97.1% respectively [37]. In another study, anti-CarP sensitivity and specificity were 44% and 89% respectively with an AUC of 0.67 [38] which was close to the present result. Similarly, in an Italian RA cohort, anti-CarP sensitivity was 46.8% and specificity 91.95% while the AUC was highest for the anti-CCP [28] as in this work. Our study found no significant association between anti-CarP antibodies and disease duration, duration of MS, TJC, SJC, VAS, ESR or CRP. Conversely, anti-CarP were significantly associated with extra-articular manifestations and deformity. In concordance, a study performed to assess the diagnostic value of anti-CarP antibodies in Iraqi patients reported absence of significant relation between anti-CarP and the duration of disease SJC, TJC, and ESR [37]. A significant relationship between anti-CarP and deformity was reported by others [39]. The anti-CarP antibodies did not associated with the disease activity in the current patients which support the findings of others [27,31,39]. In contrast, it was demonstrated that higher disease activity levels were significantly associated with anti-CarP in cases of inflammatory polyarthritis. Also, in their work, anti-CCP but not RF showed significant association with DAS28 [40]. However, that study was conducted on a large number of patients and assessed the associations between anti-CarP antibody status and longterm disease activity. This study suggested a relation of anti-CarP antibodies with poor prognosis as evidenced by the significant association between them and both radiological damage and functional disability. The relation of anti-CarP with erosions and radiological damage was also documented in other several studies [1,18,27,29,39]. More interestingly, the association of anti-CarP with radiological damage was found to be significant in anti-CCP negative patients, which may support the suggestion of the clinical usefulness of anti-CarP antibodies as a prognostic marker in anti-CCP negative patients
Table 6 Diagnostic value of different autoantibodies in rheumatoid arthritis patients. Antibody
Anti-CarP Anti-CCP RF
RA patients (n = 90) Sensitivity (95% CI)
Specificity (95% CI)
PPV
NPV
Acc.
32.2% (22.8–42.9) 61.1% (50.3–71.2) 66.7% (56.0–76.3)
96.7% (90.6–99.3) 97.8% (92.2–99.7) 91.1% (83.2–96.1)
90.6% 96.5% 82.2%
58.8% 72.5% 73.2%
64.4% 79.4% 78.9%
RA: rheumatoid arthritis, Anti-CarP: anti-carbamylated protein, Anti-CCP: anti cyclic citrullinated peptide, RF: rheumatoid factor, CI: confidence interval, PPV: positive predictive value, NPV: negative predictive value, ACC: accuracy.
Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004
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S.R. Mohamed et al. / The Egyptian Rheumatologist xxx (xxxx) xxx
and their relation to the risk of developing a more severe disease in this subgroup of RA patients [40]. In conclusion, the study confirmed the presence of anti-CarP antibodies in RA patients, and inspite of having low sensitivity; they were detected in patients negative for anti-CCP and thus could have an additive diagnostic value beside RF and anti-CCP. Anti-CarP antibodies may serve as good predictors of disease severity and are related to joint damage even in anti-CCP negative patients. More studies are required to reveal the diagnostic utility of anti-CarP antibodies especially during early stages of RA. Conflict of interest None. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Brink M, Verheul MK, Rönnelid J, Berglin E, Holmdahl R, Toes RE, et al. Anticarbamylated protein antibodies in the pre-symptomatic phase of rheumatoid arthritis, their relationship with multiple anti-citrulline peptide antibodies and association with radiological damage. Arthritis Res Ther 2015;17(1):25. [2] Hegazy MK, Auf FA, Neseem NO, Al-Harrass MF. Toll-like receptor (TLR9) -1486 T/C (rs187084) gene polymorphism in Egyptian patients with rheumatoid arthritis. The Egyptian Rheumatologist. 2018;epub ahead of print. [3] Shaker OG, El-Demellawy HH, Salem MN, Eesa NN. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in rheumatoid arthritis patients: Correlation with serum osteopontin levels and disease activity. Egyptian Rheumatologist 2016;38(4):283–8. [4] Ayeldeen G, Nassar Y, Ahmed H, Shaker O, Gheita T. Possible use of miRNAs146a and -499 expression and their polymorphisms as diagnostic markers for rheumatoid arthritis. Mol Cell Biochem 2018 Dec;449(1–2):145–56. [5] Trouw LA, Huizinga TW, Toes RE. Autoimmunity in rheumatoid arthritis: different antigens-common principles. Ann Rheum Dis 2013;72:ii132-6. [6] Allam SI, Sallam RA, Elghannam DM, El-Ghaweet AI. Clinical significance of serum B cell chemokine (CXCL13) in early rheumatoid arthritis patients. The Egyptian Rheumatologist 2019;41(1):11–4. [7] Hameed AK, El-Said TO, Askar HY, ElKady BA. Performance of serum adenosine deaminase in measuring disease activity in rheumatoid arthritis patients. The Egyptian Rheumatologist. 2018;epub ahead of print. [8] El Defrawy AO, Gheita TA, Raslan HM, El Ansary MM, El Awar AH. Serum and synovial cartilage oligomeric matrix protein levels in early and established rheumatoid arthritis. Z Rheumatol 2016;75(9):917–23. [9] Gheita TA, Sayed S, Gheita HA, Kenawy SA. Vitamin D status in rheumatoid arthritis patients: relation to clinical manifestations, disease activity, quality of life and fibromyalgia syndrome. Int J Rheum Dis 2016;19(3):294–9. [10] Gheita TA, Kenawy SA, El Sisi RW, Gheita HA, Khalil H. Subclinical reduced G6PD activity in rheumatoid arthritis and Sjögren’s Syndrome patients: relation to clinical characteristics, disease activity and metabolic syndrome. Mod Rheumatol 2014;24(4):612–7. [11] Hussein N, Kamel N, Fouda N, Farrag D. Peptidyl-arginine deiminase-type IV as a diagnostic and prognostic marker in rheumatoid arthritis patients. The Egyptian Rheumatologist. 2018; epub ahead of print. [12] Soliman AF, Egaila SE, Ali AI, Azab NI, Al-Gohary HH. HLA-DRB1 alleles in Egyptian rheumatoid arthritis patients: Relations to anti-cyclic citrullinated peptide antibodies, disease activity and severity. Egyptian Rheumatologist 2016;38(4):269–75. [13] Abdel-Magied RA, AbuOmar HA, Ali LH, Talaat H, Mohamed FI. Diagnostic potential of ultrasound in systemic lupus erythematosus patients with joint involvement: Relation to anticyclic citrullinated peptide (anti-CCP), disease activity and functional status. The Egyptian Rheumatologist. 2018;epub ahead of print. [14] El-Shazly RI, Hussein SA, Raslan HZ, Elgogary AA. Anti-mutated citrullinated vimentin antibodies in rheumatoid arthritis patients: Relation to disease activity and manifestations. Egyptian Rheumatologist 2014;36(2):65–70. [15] El-Fedawy S, Ghareeb H, Lotfy N, El-Azizi NO, Mahmoud A. Role of antimutated citrullinated vimentin antibodies in chronic hepatitis C patients and its relation to HCV associated arthritis. Egyptian Rheumatologist 2017;39 (3):175–8. [16] Pruijn GJ. Citrullination and carbamylation in the pathophysiology of rheumatoid arthritis. Front Immunol 2015;6:192.
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Please cite this article as: S. R. Mohamed, N. O. Neseem, S. S. Metwally et al., Diagnostic value and clinical significance of anti-carbamylated protein (antiCarP) antibodies in Egyptian patients with rheumatoid arthritis, The Egyptian Rheumatologist, https://doi.org/10.1016/j.ejr.2019.04.004