Introduction
Materials and methods
Patients
Autoantibody analysis
Autoantibody against | Findings classifying patients as antibody-positive | Usual additional findings |
---|---|---|
Centromere | Centromeric immunofluorescence pattern on HEp-2 cells (308 of 310 were positive) or a CENP-B band in line assay (309 of 310 positive) | |
Topoisomerase I | At least two of three findings: Scl-70-positive signal in line assay (258 of 260 positive), a band comigrating with a prototype band in IP (all positive), a line of identity in ID with the Scl-70 prototype serum (244 of 260 positive) | Typical HEp-2 cell immunofluorescence pattern: fine granular karyoplasmic, weakly nucleolar, metaphase chromosome-positive |
RNA polymerases | Characteristic IP pattern comigrating with the pattern of a prototype serum mainly consisting of four bands: Ia, Ib, IIIa and IIIb [58] | Confirmation by ELISA in 32 of 32 cases with the immunodominant epitope of RNA polymerase III subunit RPC155 according to Kuwana et al. [71], provided by Matritec, Freiburg, Germany. ANA immunofluorescence on HEp-2 cells was predominantly fine granular only sometimes (five cases) in addition nucleolar [72]. |
Fibrillarin | An IP band (approximately 34 kDa) comigrating with a prototype serum band, plus a nucleolar immunofluorescence pattern on HEp-2 cells | Confirmation by investigational ELISA kindly provided by Euroimmun, Lübeck, Germany, positive in 11 and borderline in 1 of the 12 cases |
To | An IP band of approximately 40 kDa plus a nucleolar immunofluorescence pattern on HEp-2 cells; confirmation by immunoblot analysis with recombinant To antigen kindly supplied by Dr M Blüthner, Labor Seelig, Karlsruhe, Germany | |
PM-Scl | A line of identity in ID with a PM-Scl prototype serum (41 of 42 cases) and/or positive result of ELISA with the synthetic peptide PM-1α [73] (Dr Fooke Laboratorien GmbH, Neuss, Germany) (12 of 13 cases) | Positive reaction in 37 of 41 cases for PM-Scl by line assay. ANA immunofluorescence on HEp-2 cells usually was nucleolar plus fine granular karyoplasmic. |
Ku | Two prominent IP bands at about 70 and 80 kDa comigrating with prototype bands | In 3 of 10 cases, a line identical to a Ku prototype band in ID. ANA immunofluorescence was finely granular, usually at a high titre. |
U1-RNP | A positive signal for RNP/Sm in line assay, with or without a positive signal for Sm, plus a typical IP pattern consisting of at least antigen A (about 33 kDa), antigen B/B' (about 28/29 kDa) and antigen C (about 22 kDa) | In 37 of 41 cases, a line of identity with a U1-RNP prototype in ID. ANA pattern on HEp-2 cells usually was coarsely speckled. |
Sm | A positive signal for RNP/Sm as well as for Sm in line assay | In two of four cases, a band identical to a Sm prototype in ID with ribonuclease-digested calf thymus extract. IP and immunofluorescence patterns were similar to those found for anti-U1-RNP. |
Jo-1 | A positive signal for Jo-1 in line assay plus a band identical to a Jo-1 prototype band in ID | Immunofluorescence on HEp-2 cells was inconsistent. |
Pl-7 | IP band of about 80 kDa comigrating with prototype band plus a band identical to a Pl-7 prototype band in ID | Cytoplasmic immunofluorescence on HEp-2 cells |
OJ | Typical triplet band in IP comigrating with prototype bands | Cytoplasmic immunofluorescence on HEp-2 cells |
An RNP-like IP pattern and coarsely speckled ANA immunofluorescence, without any U1-RNP signals in line assay and ID; U11-RNP specificity detected by C Will and R Lührmann, Marburg, Germany | ||
Doublet IP bands of about 25 and 23 kDa, with the 25 kDa band comigrating with the precipitate of rabbit anti-p25 kindly provided by E Chan, Gainesville, FL, USA | HEp-2 cell immunofluorescence pattern was always centromeric because anti-p25/p23 was exclusively found together with anticentromere. | |
SL | A band identical to the SL prototype band in ID plus an IP band at about 31 kDa comigrating with the precipitate of the SL reference serum | HEp-2 cell immunofluorescence pattern was fine granular, but in this study often was masked because of other coexisting antibodies |
NOR-90 | Doublet IP bands at about 90 kDa comigrating with the precipitate of a NOR-90 reference serum [78] | The nucleolar immunofluorescence pattern expected on HEp-2 cells was hard to detect in the sera examined in this study, because NOR-90 antibodies in all cases coincided with other autoantibodies visible on HEp-2 cells. |
Mitochondrial M2 | AMA M2-positive signal by line assay (40 of 41 positive) and/or AMA typical cytoplasmic immunofluorescence on HEp-2 cells and/or rat kidney sections (27 of 41 positive) | In IP, a band of around 70 kDa was present in 36 of 41 cases. |
Sp100 | Multiple nuclear dot pattern on HEp-2 cells [79] plus Sp100 signal in the line assay HUMAN IMTEC-Liver Line Immunoassay (HUMAN Diagnostics GmbH, Wiesbaden, Germany) | |
Ro52 | Ro52-positive signal by line assay | |
Ro60 | Ro60-positive signal by line assay | |
La | La-positive signal by line assay |
Statistics
Results and discussion
Autoantibodies | Patients, n(%) |
---|---|
Positive for antinuclear antibodies | 813 (94.2) |
Antibodies highly associated with SSc or scleroderma overlap syndromes | 704 (81.6) |
Anti-centromere | 310 (35.9) |
Anti-topoisomerase I | 260 (30.1) |
Anti-PM-Scl | 42 (4.9) |
Anti-U1-RNP | 41 (4.8) |
Anti-RNA polymerase | 33 (3.8) |
Anti-fibrillarin | 12 (1.4) |
Anti-To | 2 (0.2) |
Anti-Ku | 10 (1.2) |
Anti-Jo-1/-Pl-7/-OJ | 4 (0.5) |
Anti-U11-RNP | 1 (0.1) |
Other autoantibodies | |
Anti-Ro and/or anti-La | 206 (23.9) |
Anti-Ro52 | 187 (21.7) |
Anti-Ro60 | 59 (6.8) |
Anti-La | 16 (1.9) |
Anti-mitochondrial M2 | 41 (4.8) |
Anti-p25/p23 | 28 (3.2) |
Anti-NOR-90 | 6 (0.7) |
Anti-SL | 9 (1.0) |
Anti-Sm | 4 (0.5) |
Anti-Sp100 | 4 (0.5) |
Other (known or unknown) | 363 (42.1) |
Negative for all highly SSc-associated antibodies | 159 (18.4) |
Negative for antinuclear antibodies | 50 (5.8) |
Autoantibody-negative by all criteria used | 38 (4.4) |
Antibodies | ACA | ATA | Anti-PM-Scl | Anti-U1-RNP | Anti-RNAP | Anti-fibrilla-rin | Anti-To | Anti-Ku | Anti-Jo-1/Pl-7/OJ | Anti-U11-RNP | Anti-Ro52 | Anti-Ro60 | Anti-La | AMA | Anti-p25/p23 | Anti-NOR-90 | Anti-SL | Anti-Sm | Anti-Sp100 | Other |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ACA | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 92 | 11 | 1 | 31 | 28 | 4 | 2 | 0 | 3 | 144 | |
ATA | 1 | 2 | 0 | 0 | 0 | 4 | 0 | 0 | 36 | 26 | 6 | 4 | 0 | 1 | 4 | 1 | 0 | 77 | ||
Anti-PM-Scl | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 6 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | |||
Anti-U1-RNP | 1 | 0 | 0 | 0 | 0 | 0 | 11 | 6 | 1 | 2 | 0 | 1 | 0 | 4 | 0 | 11 | ||||
Anti-RNAP | 0 | 0 | 0 | 0 | 0 | 5 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 5 | |||||
Anti-fibrillarin | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 8 | ||||||
Anti-To | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | |||||||
Anti-Ku | 0 | 0 | 2 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 2 | ||||||||
Anti-Jo-1/Pl-7/OJ | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |||||||||
Anti-U11-RNP | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||||||||
Anti-Ro52 | 41 | 15 | 14 | 11 | 2 | 5 | 2 | 2 | 97 | |||||||||||
Anti-Ro60 | 14 | 6 | 0 | 0 | 0 | 1 | 1 | 33 | ||||||||||||
Anti-La | 1 | 0 | 0 | 0 | 1 | 0 | 8 | |||||||||||||
AMA | 2 | 0 | 1 | 1 | 2 | 29 | ||||||||||||||
Anti-p25/p23 | 1 | 1 | 0 | 1 | 18 | |||||||||||||||
Anti-NOR-90 | 0 | 1 | 0 | 4 | ||||||||||||||||
Anti-SL | 0 | 0 | 3 | |||||||||||||||||
Anti-Sm | 0 | 0 | ||||||||||||||||||
Anti-Sp100 | 2 | |||||||||||||||||||
Other | ||||||||||||||||||||
Isolateda | 104 | 153 | 31 | 19 | 23 | 4 | 0 | 4 | 1 | 1 | 2 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 79 |
Totalb | 310 | 260 | 42 | 41 | 33 | 12 | 2 | 10 | 4 | 1 | 187 | 59 | 16 | 41 | 28 | 6 | 9 | 4 | 4 | 363 |
Limited (N= 513) | Diffuse (N= 173) | Overlap (N= 108) | Undifferentiated (N= 64) | ||||
---|---|---|---|---|---|---|---|
Antibodies | n(%) | ORa (P-value) | n(%) | OR (P-value) | n(%) | OR (P-value) | n(%) |
ACA |
253 (49.3)
|
5.00 (
P
< 0.0001)
| 12 (6.9) | 16 (14.8) | 28 (43.8) | ||
ATA | 141 (27.5) |
98 (56.6)
|
4.26 (
P
< 0.0001)
| 11 (10.2) | 9 (14.1) | ||
Anti-RNAP | 14 (2.7) |
14 (8.1)
|
3.11 (
P
= 0.0029)
| 2 (1.9) | 2 (3.1) | ||
Anti-U1-RNP | 7 (1.4) | 0 (0.0) |
31 (28.7)
|
30.00 (
P
< 0.0001)
| 2 (3.1) | ||
Anti-PM-Scl | 16 (3.1) | 2 (1.2) |
22 (20.4)
|
9.40 (
P
< 0.0001)
| 2 (3.1) | ||
Anti-fibrillarin | 3 (0.6) |
8 (4.6)
|
8.32 (
P
= 0.0005)
| 1 (0.9) | 0 (0.0) | ||
Anti-To | 1 (0.2) | 0 (0) | 1 (0.9) | 0 (0.0) | |||
Anti-Jo-1/Pl-7/OJ | 0 (0.0) | 0 (0) |
4 (3.7)
|
65.07 (
P
= 0.0002)
| 0 (0.0) | ||
Anti-U11-RNP | 0 (0.0) | 1 (0.6) | 0 (0.0) | 0 (0.0) | |||
Anti-Ku | 5 (1.0) | 1 (0.6) | 3 (2.8) | 1 (1.6) | |||
Anti-SL | 5 (1.0) | 3 (1.7) | 1 (0.9) | 0 (0.0) | |||
Anti-Sm | 0 (0.0) | 0 (0.0) | 3 (2.8) | 21.54 (P = 0.0069) | 0 (0.0) | ||
Anti-NOR-90 | 5 (1.0) | 0 (0.0) | 1 (0.9) | 0 (0.0) | |||
AMA | 28 (5.5) | 4 (2.3) | 4 (3.7) | 5 (7.8) | |||
Anti-Sp100 | 3 (0.6) | 0 (0.0) | 0 (0.0) | 1 (1.6) | |||
Anti-Ro52 | 125 (24.4) | 1.50 (P = 0.023) | 20 (11.6) | 27 (25.0) | 15 (23.4) | ||
Anti-Ro60 | 28 (5.4) | 14 (8.1) | 13 (12.0) | 2.11 (P = 0.0382) | 4 (6.3) | ||
Anti-La | 9 (1.8) | 3 (1.7) | 1 (0.9) | 3 (4.7) | |||
Anti-p25/p23 |
24 (4.5)
|
4.25 (
P
= 0.0031)
| 0 (0.0) | 2 (1.9) | 2 (3.1) | ||
Other | 209 (40.7) | 74 (42.8) | 44 (40.7) | 34 (53.1) | |||
ANA-negative | 32 (6.2) | 6 (3.5) | 5 (4.6) | 7 (10.9) |
ACA (310) | ATA (260) | Anti-PM-Scl (42) | Anti-U1-RNP (41) | Anti-RNA -P (33) | Anti-Fibrillarin (12) | Anti-Ku (10) | Anti-Ro52 (187) | Anti-Ro60 (59) | Anti-La (16) | AMA (41) | Anti-p25/23 (28) | ANA-negative (50) | no SSc associated ab's (161) | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
male sex 148 (17.1%) |
17
0.19 (0.11 to 0.32)
P < 0.0001 |
64
2.02 (1.4 to 2.91)
p = 0.0002 | 6 | 9 | 8 | 4 | 2 | 23 0.62 (0.38 to 0.997 p = 0.049 | 12 | 2 | 1 | 1 | 10 |
41
1.90 (1.261 to 2.863)
p = 0,0035 |
age at disease onset < 50 y 399/781 (51.1%) |
115
0.57 (0.43 to 0.77)
p = 0.0003 | 135 1.39 (1.02 to 1.88) p = 0.0431 | 22 |
29
2.92 (1.40 to 6.07)
p = 0.0029 | 13 | 8 | 4 | 77 | 31 | 10 | 20 | 8 | 18 | 73 |
Rodnan skin score > 10 294/750 (39.2%) |
58
0.27 (0.19 to 0.38)
P < 0.0001 |
132
3.10 (2.24 to 4.27)
P < 0.0001 | 14 | 9 |
18
3.24 (1.44 to 7.31)
p = 0.0042 | 7 | 1 | 58 | 23 | 6 | 10 | 11 | 14 | 56 |
Raynaud's phenomenon 819 (94.9%) | 301 2.26 (1.07 to 4.77) p = 0.0349 | 252 | 37 | 41 | 32 | 12 | 10 | 184 3.96 (1.2 to 12.94 p = 0.0133 | 58 | 16 | 41 | 28 | 43 0.29 (0.12 to 0.70) p = 0.0104 |
140
0.23 (0.12 to 0.42)
P < 0.0001 |
Digital ulcers 216/840 (25.7%) |
55
0.50 (0.36 to 0.71)
P < 0.0001 |
106
3.18 (2.30 to 4.41)
P < 0.0001 | 9 | 11 | 7 | 4 | 1 | 48 | 19 | 6 | 13 | 6 | 4 0.26 (0.09 to 0.76) p = 0.0076 | 28 0.60 (0.39 to 0.94) p = 0.024 |
Pulmonary hypertension 126 (14.6%) | 57 1.58 (1.08 to 2.32) p = 0.0208 | 36 | 2 | 9 | 4 | 1 | 0 | 31 | 14 | 2 | 2 | 7 | 2 0.23 (0.06 to 0.97) p = 0.0232 | 17 |
Pulmonary fibrosis 287 (33.3%) |
39
0.18 (0.12 to 0.26)
P < 0.0001 |
151
4.76 (3.48 to 6.50)
P < 0.0001 | 16 | 11 | 7 | 2 | 6 | 69 |
30
2.20 (1.29 to 3.75
p = 0.0040 | 9 | 6 0.33 (0.14 to 0.79) p = 0.01 | 4 0.33 (0.11 to 0.95) p = 0.0393 | 11 | 55 |
Lung restrictive defect 218/833 (26.2%) |
41
0.31 (0.21 to 0.45)
P < 0.0001 |
104
2.96 (2.14 to 4.09)
P < 0.0001 | 9 | 7 | 8 | 3 | 4 | 45 | 19 | 4 | 8 | 4 | 10 | 34 |
Esophageal involvement 535 (62.0%) | 198 | 175 1.39 (1.02 to 1.89) p = 0.039 |
14
0.29 (0.152 to 0.56)
p = 0.0001 | 29 | 20 | 7 | 6 | 120 | 39 | 13 | 24 | 21 | 27 | 87 0.67 (0.47 to 0.94) p = 0.0243 |
Proteinuria 90/830 (10.8%) | 23 0.56 (0.34 to 0.92) p = 0.0207 | 34 | 4 | 4 | 4 | 1 | 1 | 15 | 9 | 2 | 3 | 1 | 5 | 19 |
Cardiac involvement 114 (13.2%) |
27
0.51 (0.32 to 0.81)
p = 0.0033 | 45 1.62 (1.08 to 2.44) p = 0.0216 | 5 | 7 | 5 | 1 | 2 | 20 | 9 | 1 | 4 | 1 | 5 | 22 |
Musculoskeletal involvement 421/852 (49.4%) |
131
0.64 (0.48 to 0.85)
p = 0.0022 | 130 | 20 | 28 2.49 (1.25 to 4.96) p = 0.009 | 17 | 10 5.22 (1.14 to 23.97) p = 0.0202 | 6 | 80 0.71 (0.51 to 0.99) p = 0.0468 | 32 | 12 3.13 (1.002 to 9.79) p = 0.0447 | 17 | 9 | 25 | 79 |
Synovitis 157/837 (18.8%) |
35
0.43 (0.29 to 0.65)
P < 0.0001 |
62
1.71 (1.19 to 2.45)
p = 0.0049 | 8 | 13 2.27 (1.14 to 4.53) p = 0.0326 | 8 | 2 | 2 | 32 | 15 | 5 | 2 0.21 (0.05 to 0.89) p = 0.0216 | 2 | 10 | 29 |
Joint contractures 253/840 (30.1%) |
54
0.36 (0.25 to 0.50)
P < 0.0001 |
107
2.26 (1.65 to 3.08)
P < 0.0001 | 9 | 9 | 10 | 6 | 2 | 49 | 23 | 6 | 9 | 9 | 20 1.93 (1.05 to 3.54) p = 0.0436 | 56 |
Tendon friction rubs 88/840 (10.5%) |
14
0.30 (0.16 to 0.53)
P < 0.0001 | 32 | 2 | 5 | 6 | 3 | 1 | 21 | 5 | 2 | 2 | 0 | 6 | 25 1.95 (1.18 to 3.22) p = 0.0122 |
CK elevation 74/835 (8.9%) |
14
0.37 (0.21 to 0.68)
p = 0.0009 | 20 |
19
3.56 (1.67 to 7.57)
p = 0.0023 | 5 | 6 2.60 (1.03 to 6.55) p = 0.0485 | 0 | 3 5.32 (1.30 to 21.72) p = 0.038 | 21 | 8 | 2 | 1 | 0 | 6 | 16 |
Sicca syndrome 366/858 (42.7%) | 150 1.44 (1.19 to 6.61) p = 0.0119 | 98 | 14 | 18 | 12 | 5 | 4 | 96 1.57 (1.13 to 2.17) p = 0.0075 | 33 1.85 (1.08 to 3.17) p = 0.0275 | 12 4.14 (1.32 to 12.93) p = 0.0102 | 19 |
20
3.50 (1.52 to 8.03)
p = 0.0029 | 23 | 66 |
Mouth involvement 223/829 (26.9%) |
64
0.61 (0.44 to 0.85)
p = 0.0034 |
93
2.1 (1.53 to 2.93)
P < 0.0001 | 10 | 7 | 10 | 3 | 4 | 49 | 18 | 6 | 11 | 6 | 12 | 34 |
ESR > 25 mm/h 199/741 (26.9%) | 58 0.70 (0.49 to 0.99) p = 0.046 | 75 1.55 (1.10 to 2.19) p = 0.015 | 4 0.33 (0.11 to 0.94) p = 0.0325 | 14 | 5 | 0 | 0 |
60
1.76 (1.21 to 2.54)
p = 0.0039 |
28
3.39 (1.92 to 5.97)
P < 0.0001 | 9 3.62 (1.33 to 9.86) p = 0.0447 | 10 | 5 | 15 | 42 |
Quantitative traits | Clinical data | ||
---|---|---|---|
Age at disease onset |
n
| Mean ± SD (years) | P* |
Total | 781 | 47.7 (14.2) | |
Anti-centromere | 273 | 51.3 (12.5) | < 0.0001 |
Anti-topoisomerase I | 238 | 46.0 (14.0) | 0.0076 |
Anti-fibrillarin | 12 | 38.8 (16.0) | 0.0404 |
Anti-U1-RNP | 39 | 38.2 (15.0) | < 0.0001 |
Anti-La | 15 | 37.9 (18.1) | 0.0431 |
Autoantibody-negative | 36 | 52.9 (14.7) | 0.0205 |
Rodnan skin score |
n
| Mean score ± SD |
P
|
Total | 750 | 10.2 (9.4) | |
Anti-centromere | 275 | 6.4 (6.0) | < 0.0001 |
Anti-topoisomerase I | 227 | 14.1 (9.7) | < 0.0001 |
Anti-RNA polymerase | 27 | 15.7 (11.7) | 0.0091 |
Anti-fibrillarin | 10 | 21.2 (15.0) | 0.0108 |
Anti-U1-RNP | 35 | 6.9 (9.2) | 0.0053 |
Erythrocyte sedimentation rate |
n
| Mean ± SD (mm/hour) |
P
|
Total | 741 | 19.46 (16.7) | |
Anti-topoisomerase I | 227 | 22.95 (19.1) | 0.0002 |
Anti-PM-Scl | 36 | 12.19 (9.4) | 0.0014 |
Anti-Ro52 | 167 | 22.05 (18.8) | 0.0374 |
Anti-Ro60 | 53 | 28.47 (19.6) | < 0.0001 |
Anti-La | 16 | 28.56 (20.1) | 0.0447 |