The seroprevalence of anti-Ro52 in the SS patients
The 115 patients with SS were classified as 87 primary SS and 28 secondary SS. Among these 115 patients with SS, 70 patients (60.9%) were anti-Ro52 positive (Fig. 2A). The p value for group differences of age between the patients with SS vs healthy subjects and the patients with SS vs the patients with RA was <0.001 and 0.018, respectively. In addition, the p value for group differences of sex between the patients with SS vs healthy subjects and the patients with SS vs the patients with RA was <0.001 and 0.025, respectively. Therefore, no significant differences in the distribution of age and sex were observed among these 3 groups. There were two cases (3.2%) of positive anti-Ro52 among the 62 patients with RA, and there was no positive case among the 50 normal control subjects (Fig. 2A). The 2 secondary SS patients with ACA+SSc patients fulfilled 2013 ACR/EULAR classification criteria for SSc and had neither anti-Ro52 nor anti-Ro60. The group of 115 SS patients included 95 patients with anti-Ro60 (82.6%). There were 34 non-SS subjects who were not classified as SS by AECG criteria. In the non-SS subjects, 7 subjects were positive for anti-Ro52 (Fig. 2A).
The background characteristics of the patients with SS according to the presence or absence of anti-Ro52 are summarized (Table 1). Five items were significantly high in the SS patients with anti-Ro52. We also examined the subjects' treatment for sicca symptoms and the use of immunosuppressants. There was no case of congenital heart block (CHB) in the patients' medical records. According to their status as being with or without anti-Ro60, the SS patients with anti-Ro52 were divided 2 groups. Anti-Ro60+ SS patients included anti-Ro60+anti-Ro52+ patients (n=67), and the anti-Ro60+anti-Ro52− patients (n=28). In contrast, anti-Ro60- SS patients included the anti-Ro60-anti-Ro52+ patients (n=3), and the anti-Ro60-anti-Ro52− patients (n=17) (Fig. 2B). We also observed markedly high anti-Ro52 (≥500 U/ml) in 31 (44.3%) of the 70 anti-Ro52-positive patients with SS (Fig. 2C).
Table 1. Clinical characteristics of the SS patients
Variables at diagnosis
|
Anti-Ro52
positive
(n=70)
|
Anti-Ro52 negative
(n=45)
|
p-value
|
Age, yrs; mean±SD
|
58.8±14.6
|
58.8±14.0
|
0.92
|
Female patients, n (%)
|
68 (97.1)
|
41 (91.1)
|
0.16
|
Xerophthalmia, n (%)
|
42 (60.0)
|
33 (75.0)
|
0.07
|
Xerostomia, n (%)
|
53 (75.7)
|
39 (86.7)
|
0.12
|
Positive Saxon test, n (%)
|
54 (84.4)
|
32 (74.4)
|
0.15
|
Positive Schirmer's test, n (%)
|
41 (68.3)
|
30 (75.0)
|
0.31
|
Anti-Ro60, n (%)
|
67 (95.7)
|
28 (62.2)
|
<0.01*
|
Anti-La/SS-B antibody, n (%)
|
31 (44.3)
|
8 (17.8)
|
<0.01*
|
Positive ANA, n (%)
|
67 (95.7)
|
41 (91.1)
|
0.27
|
Positive RF, n (%)
|
50 (75.8)
|
12 (29.2)
|
<0.01*
|
Serum IgG mg/dl, mean±SD
|
1996 ± 619
|
1579 ± 461
|
<0.01*
|
WBC count, x103/µl
|
5018 ± 2434
|
5055 ± 1412
|
0.92
|
Hemoglobin, g/dl
|
12.3 ± 1.5
|
12.7 ± 1.1
|
0.04*
|
Platelet count, x103/µl
|
20.3 ± 8.6
|
22.5 ± 5.1
|
0.08
|
Focus score, mean±SD
|
4.5 ± 2.8
|
4.3 ± 3.6
|
0.78
|
ESSDAI score, mean±SD
|
2.0 ± 2.0
|
0.8 ± 2.1
|
<0.01*
|
Use of ophthalmic solutions,
n (%)
|
18 (25.7)
|
11 (24.4)
|
0.53
|
Use of muscarinic receptor agonists,
n (%)
|
25 (35.7)
|
14 (31.1)
|
0.38
|
Use of immunosuppressants, n (%)
|
15 (21.4)
|
4 (8.9)
|
0.06
|
*p<0.05. Welch's t-test or Fisher's exact test was used. Ophthalmic solutions included chondron instillation, purified sodium hyaluronate, artificial tear ophthalmic solution, rebamipide ophthalmic suspension, and diquafosol sodium. Muscarinic receptor agonists included cevimeline hydrochloride hydrate and pilocarpine hydrochloride. Immunosuppressants included oral prednisolone (PSL), tacrolimus, and mizoribine. PSL was used for 9 of the 15 anti-Ro52-positive secondary SS cases and 3 of the 4 anti-Ro52-negative secondary SS cases. ANA: anti-nuclear antibody, anti-Ro52: anti-Ro52/SS-A antibody, anti-Ro60: anti-Ro60/SS-A antibody, ESSDAI; EULAR Sjögren's Syndrome Disease Activity Index, RF: rheumatoid factor. WBC: white blood cell.
Although we identified anti-Ro52 positivity in 75% (63/84) of the anti-Ro60+ACA− patients with SS, there was only one anti-Ro52-positive case among the 16 anti-Ro60-ACA+ patients with SS (Fig. 2D); this case showed an equivocal anti-Ro52 concentration (7.44 U/ml) and was considered anti-Ro52-positive in this study. The odds ratio of anti-Ro60 positivity over anti-Ro52 positivity was 13.2 (95%CI: 3.4–76.0). The odds of anti-Ro60 being positive were significantly higher in the anti-Ro52-positive patients than in the ACA-positive patients (p<0.05, permutation test for ratio of odds ratio).
Clinical characteristics associated with relevance between anti-Ro60/Ro52
As described above, our present findings replicate the association between the positivity of anti-Ro52 and that of anti-Ro60 that had been reported previously [29, 30]. We also determined the clinical characteristics with respect to the increment of anti-Ro60 concentration toward increment of the anti-Ro52 concentration. The results of our analyses revealed that the effect of the following clinical characteristics were significant (the values in parentheses are 95%CIs of the regression coefficients of interaction terms): xerophthalmia positivity (-9.49 to -0.42), xerostomia positivity (-8.23 to -3.15), ACA positivity (-7.85 to -1.80), RP positivity (-7.45 to -1.27), high serum IgG (0.02 to 0.85), and RF positivity (1.43 to 12.24).
The relationships between the levels of the two subtypes of anti-Ro are illustrated (Figure 3). The subjects were stratified by the positivity of the respective clinical characteristics, and each subject subgroup's data were plotted separately. The figure's scatterplots reveal a blank range for anti-Ro60 (the boundaries are 4.4 and 34.9 on the x-axis) which divided the subjects into two parts (the lower-left part and the right side in the plots). The unbalanced nature of the density of values in the lower-left parts of the scatterplots among the subgroups is clear, especially for the four items in which significant downward effects of positivity on the regression coefficient were revealed; e.g., between xerophthalmia (−) and xerophthalmia (+). It is possible that the significance of the regression coefficients of the interaction terms is attributable to this clustered unbalancedness. As we noted above, it was also clear that the relationships between the levels of anti-Ro52 and anti-Ro60 were not linear, but we used a linear regression in order to grasp the characteristics of the global relationship.
The association between clinical characteristics and anti-Ro60/Ro52
We compared the concentration of anti-Ro52 and anti-Ro60 between the subgroups of subjects classified by components in the AECG criteria and by other items including RP, ACA, RF, and serum IgG (Suppl. Fig. S1). The comparison revealed that three characteristics (RP, ACA, and IgG) with which subgroups were created were significantly different according to the titer for both antibodies (p<0.05 for each characteristic). Regarding the anti-Ro52 concentration, the differences were significant in three additional characteristics: xerophthalmia, anti-La/SS-B antibody, and RF. For the anti-Ro60 concentration, there was no additional characteristic with which the association was significant aside from RP, ACA, and IgG.
We determined the maximal information coefficients to identify the clinical characteristics with significant dependency between anti-Ro52 or anti-Ro60 or both, and the results demonstrated that the dependency between anti-La/SS-B and both anti-Ro52 and anti-Ro60 was significant (p<0.05 for each antibody) (Suppl. Fig. S2). We also investigated which clinical characteristics are associated with the anti-Ro52 concentration by determining the ROC curve (Fig. 4). The characteristics in which positivity was highly discriminated by the concentration of anti-Ro52 were as follows: ACA, ESSDAI ≥1, and RF (AUC >0.75, respectively) (Fig. 4). Moderately discriminated characteristics were serum IgG, FS ≥1, and anti-La/SS-B antibody (AUC >0.70, respectively) (Fig. 4).
We analyzed the linear relationships between the ESSDAI score and levels of antibodies against the two respective subtypes of Ro/SSA antigens. The range of our subjects' ESSDAI scores was 0 to 17. In the linear regression, the patients with the ESSDAI score >4 were put together in the subgroup of 'ESSDAI ≥4,' and in this analysis, significant linearity between the ESSDAI scores and anti-Ro52 was confirmed (Fig. 5) as anticipated from the correlation with anti-Ro60. The details of the ESSDAI scores of the entire group of SS patients and of the anti-Ro52-positive SS patients are also shown (Suppl. Fig. S3), indicating the presence of a high frequency of the biological item and a low frequency of the articular item. We also displayed the level of anti-Ro52 and anti-Ro60 according to positive ESSDAI domain (Suppl. Fig. S4 and S5), indicating that the level of anti-Ro52 in all domains that had positive number showed more than cutoff value, 10 U/ml.
To determine the influence of secondary SS, we examined the clinical characteristics that were relevant to the association between the anti-Ro52 and anti-Ro60 concentration in primary SS by deleting the data of the 28 patients with secondary SS, and we obtained the following significant findings: xerostomia positivity (-8.24 to -3.19), ACA positivity (-7.84 to -1.77), RP positivity (-7.48 to -1.18), high serum IgG (0.04 to 0.86), RF positivity (1.34 to12.10) (the values in the parentheses are 95%CIs of the regression coefficients of interaction terms). Compared to the findings of total SS in Figure 3, the significance of xerophthalmia (−9.56 to 0.90) disappeared (Suppl. Fig. S6).