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Autoantibody Reactive with RNA Polymerase III in Systemic Sclerosis

Yutaka Okano, MD; Virginia D. Steen, MD; and Thomas A. Medsger, MD
[+] Article and Author Information

From the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Requests for Reprints: Thomas A. Medsger, Jr., MD, Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Pittsburgh School of Medicine, 985 Scaife Hall, Pittsburgh, PA 15261. Acknowledgments: The authors thank Drs. R. Kovelman and R. G. Roeder (Rockefeller University School of Medicine) for providing the partially purified RNA polymerase III; Dr. M. Schmidt (University of Pittsburgh School of Medicine) for providing HeLa S100 extracts and pVA I DNA template; Colleen Thomas, Claudia Conte, and Noreen Fertig for providing technical assistance; and Joan Neitznick and Roberta Shope for secretarial assistance. Grant Support: In part by The Arthritis Foundation, Western Pennsylvania Chapter (Shoemaker Fund), Pittsburgh, Pennsylvania; The Scleroderma Federation, New York, New York; The RGK Foundation, Austin, Texas; National Institutes of Health grant 5M01RR00056; and the Arthritis, Rheumatism and Aging Medical Information System (ARAMIS) National Institutes of Health grant AR21393.


Copyright 2004 by the American College of Physicians


Ann Intern Med. 1993;119(10):1005-1013. doi:10.7326/0003-4819-119-10-199311150-00007
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Objective: To determine the clinical significance of anti-RNA polymerase III antibody in systemic sclerosis (SSc).

Design: A point prevalence study of autoantibody to RNA polymerase III and longitudinal examination of its clinical significance in patients with SSc and in controls.

Setting: University medical center rheumatology practice.

Patients: Two hundred fifty-two consecutive new patients with SSc and 170 controls (150 patients with other connective tissue diseases and 20 normal volunteers).

Measurements: The presence of anti-RNA polymerase III antibody was determined by immunoprecipitation, immunoblotting, and immunodepletion studies.

Main Results: Serum specimens from 57 of the 252 patients with SSc (23%; 95% CI, 18% to 28%) reacted with RNA polymerase III, compared with none of the specimens from 170 controls (0%; 95% CI, 0% to 2%). In 40 of these 57 specimens, immunoprecipitation studies also showed the presence of RNA polymerase I or II, or both. Anti-RNA polymerase III antibody was detected in sera from 50 of the 111 patients (45%) who had SSc with diffuse cutaneous involvement (dcSSc), 7 of 114 patients (6%) who had SSc with limited cutaneous involvement, and none of 27 patients with an SSc overlap syndrome (P < 0.001). Among patients with dcSSc, anti-RNA polymerase III antibody was more common than antitopoisomerase I antibody (45% compared with 27%; P = 0.008). Patients with anti-RNA polymerase III antibody had a statistically significant higher mean maximum skin thickness score but statistically significant lower frequencies of telangiectasias, inflammatory myopathy, restrictive lung disease, and serious cardiac abnormalities than did patients with antitopoisomerase I antibody.

Conclusion: Anti-RNA polymerase III antibody is a new marker autoantibody for many patients who have SSc with diffuse or extensive cutaneous involvement.

Figures

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Figure 1.
Precipitated proteins of [35S]methionine-labeled HeLa cell extract obtained using serum specimens and fractionated on 7.leftright

5% ( ) and 12% ( ) polyacrylamide-SDS gel. Lane Mr shows molecular weight markers; lane 1 shows nonspecific proteins precipitated by normal human serum; lanes 2, 3, 4, 5, 6, and 7 show specific proteins precipitated in serum specimens from six patients with systemic sclerosis. The positions of molecular weight standards (in kilodaltons) are shown to the left of lane Mr. Two panels are included to show the separation of proteins with molecular weights of more than 30 kd and less than 40 kd, respectively.

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Figure 2.
Serum antibodies from patients with SSc shown by immunoblotting to be reactive with HeLa cell RNA polymerase III.9Table 1[21]

Each lane contained partially purified RNA polymerase III obtained from 5 10 HeLa cells by biochemical procedures. The substrate was fractionated on a 11% polyacrylamide-SDS gel and electrophoretically transferred to nitrocellulose sheets. The sheets were incubated with normal serum (lane 1) and four representative serum specimens from groups 1, 2, and 3 (lanes 2-5); the groups are defined in . The IgG bound to proteins was visualized using enzyme-linked immunoassay. Dots beside lanes indicate the positive protein bands. The nitrocellulose strip was also stained with amido-black (lane 6). An arrow to the right of lane 6 indicates the position of each subunit protein, according to the conformity of its molecular weight, as previously described . Four arrowheads to the right of lane 6 indicate the positions of four additional proteins, which are likely contaminants. The positions of molecular weight standards (in kilodaltons) are given to the right of the panel.

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Figure 3.
Depletion of RNA polymerase III activity from HeLa S100 extracts by immunoprecipitation.Table 2323

Antibodies were coupled to protein-A sepharose and used in depletion experiments. The HeLa S100 extract was preincubated with IgG-coupled protein-A sepharose, obtained using normal human serum (lane 1), four control sera with various antinuclear antibodies (the specificities are marked at the top of lanes), and six representative serum specimens from the three groups (lanes 4-9); see for group classification. We also carried out mock immunoprecipitation (no antibodies coupled to the protein-A sepharose). After removal of the sepharose by brief centrifugation, the supernatant was subjected to the RNA polymerase III transcription reactions using adenovirus pVA I as the DNA template. The [ P]orthophosphate-labeled adenovirus VA I RNA products by the in vitro transcription reactions were determined by 8% polyacrylamide-urea (7 mol/L) gels and visualized by autoradiography. Anti-topo I = anti-topoisomerase I antibody I; anti-U3 RNP = anti-U ribonucleoprotein antibody (fibrillarin).

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Figure 4.
Indirect immunofluorescence test for antinuclear antibody using HEp-2 cells as substrate.Top.[16]Bottom.

Serum specimens from patients with systemic sclerosis were diluted 1:40 with phosphate-buffered saline and used as the first antibody. Immunofluorescence shows nucleolar staining, suggesting that serum contains antinuclear antibody that is apparently identical to the anti-RNA polymerase I antibody shown with antinucleolar staining by Reimer and colleagues . Immunofluorescence shows nuclear speckled staining without nucleolar staining.

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