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Clinical Relevance of Measurement of Antibodies to Individual snU^Sub 1^-RNP Proteins

Posted on: Friday, 7 October 2005, 03:01 CDT

By Luyckx, Ariane; Westhovens, Ren; Oris, Els; Papisch, Wolfgang; Bossuyt, Xavier

Anti-ribonucleoprotein (RNP) antibodies are found in mixed connective tissue disease (MCTD), a syndrome characterized by features of systemic lupus erythematosus (SLE), inflammatory muscle disease, and scleroderma (1). High titers of anti-RNP antibodies support the diagnosis of MCTD, and testing should be ordered when the diagnosis is suspected (2). Anti-RNP antibodies are also found in rheumatic diseases such as SLE, Sjgren syndrome, rheumatoid arthritis, polymyositis, and systemic sclerosis (2).

Table 1. Association between autoantibodies and clinical symptoms.a

The antigens to which anti-RNP antibodies react reside in many proteins (70 kD, protein A, and protein C) complexed with small nuclear U^sub 1^-RNA. This complex is called small nuclear U^sub 1^ ribonucleoprotein (snU^sub 1^-RNP). Similarly, the antigen to which anti-Sm antibodies bind is composed of a complex of proteins (B, B', D, E, F, and G) and snRNAs (U^sub 1^, U^sub 2^, U^sub 4^-U^sub 6^, and U^sub 5^). Anti-Sm antibodies are highly specific for SLE (2). The snRNPs (U^sub 1^-RNP/Sm) are involved in the splicing of precursor messenger RNA.

Traditionally, anti-RNP antibodies have been detected by techniques such as passive hemagglutination, immunodiffusion, counterimmunoelectrophoresis, and ELISA using purified antigen. More recently, recombinant antigens have been used increasingly to identify anti-RNP antibodies, thus allowing identification of antibodies to the individual proteins of the snU^sub 1^-RNP complex (70 kD, protein A, and protein C) (3).

In the present study, we investigated whether the presence of antibodies to individual proteins of the snU^sub 1^-RNP complex is associated with clinical symptoms and/or organ involvement in SLE patients and patients with MCTD. We collected 37 consecutive serum samples obtained from different individuals in which anti-RNP antibodies were identified by dot blot with a purified antigen (BMD) and measured antibodies to recombinant RNP-A, RNP-C, and RNP-70 kD by use of Varelisa Split ANA Profile (Pharmacia Diagnostics). The source population was university hospital-based, and the patients had symptoms at the time anti-RNP testing was ordered. Each sample corresponded to one individual.

Of the 37 anti-RNP-positive samples, 14 were from patients with SLE and 20 from individuals with MCTD. SLE was diagnosed according to the American College of Rheumatology classification criteria revised in 1997 (3), and MCTD was diagnosed according to the criteria proposed by Alercn-Segovia et al. (4). One patient had chronic idiopathic urticaria, 1 patient had rheumatoid arthritis, and 1 had bacterial parotitis. None of these 3 individuals had signs of systemic disease. The clinical symptoms and organ involvement of the patients are summarized in Table 1. Raynaud phenomenon was present in 18 (90%) of the 20 MCTD patients and in 10 (70%) of the 14 SLE patients, consistent with the previously reported association of anti-RNP antibodies and Raynaud phenomenon (5). In both diseases, arthritis was present in 50%-55% of the patients. Lymphopenia was present in 11 (78%) of the 14 SLE patients. Glomerulonephritis and lymphopenia were found mainly in SLE patients, whereas polymyositis, esophageal dysmotility, and scleroderma were found mainly in MCTD patients. In one sample (obtained from a SLE patient), no antibodies to recombinant protein A, protein C, and the 70 kD protein could be identified. In this patient, anti-RNP antibodies were revealed only when a purified antigen was used.

Antibodies to (a) RNP-A, RNP-C, and RNP-70 kD; (b) RNP-A and RNP- 70 kD; (c) RNP-A and RNP-C; (d) RNP-C and RNP-70 kD; (e) RNP-A only; (f) RNP-C only; and (g) RNP-70 kD only were found in, respectively, 9, 0, 3, 1, 0, 0, and 0 of the 14 SLE patients and in 13, 2, 2, 1, 0, 0, and 2 of the 20 MCTD patients. These distributions were comparable in SLE and MCTD. The patients with chronic idiopathic urticaria, rheumatoid arthritis, or parotitis had antibodies to, respectively, RNP-70 kD only, RNP-A and RNP-C, and RNP-70 kD only. Table 1 contains a survey of the symptoms and the antibodies to the individual snU^sub 1^-RNP proteins in patients with anti-RNP antibodies. The statistical significance of associations between autoantibodies and symptoms was explored by Fisher 2-tailed exact test. Within the patient group with anti-RNP antibodies, patients with a specific combination of antibodies were compared with all other patients. This was done for each symptom and for each specific combination of antibodies. No correction for multiple testing was made, but only a P value <0.01 was taken to indicate significance.

We found no associations between antibodies to the individual snU^sub 1^-RNP proteins and symptoms or organ involvement except for an association between decreased lung diffusion and the presence of antibodies to RNP-A as well as to RNP-C (in the absence of antibodies to RNP-70 kD; P = 0.0068). Antibodies to proteins A and C were present in 4 of the 7 patients with anti-RNP antibodies and decreased lung diffusion but were present in only 2 of the 30 individuals with anti-RNP antibodies but without decreased lung diffusion. Such an association needs to be confirmed in future studies.

In addition to antibodies to RNP-A, RNP-C, and RNP-70 kD, we also measured antibodies to recombinant Sm-B/B' (Varelisa Split ANA Profile) and to purified Sm-D (EHa-Sm; Pharmacia Diagnostics) in the 37 samples with anti-RNP antibodies. Anti-Sm-D antibodies were found in 5 of the 14 SLE patients and in none of the MCTD patients (P = 0.0046). Anti-Sm-B/B' antibodies were found in 9 of the 14 SLE patients and in 11 of the 20 MCTD patients (P = 0.728). Four of the 5 patients with anti-Sm-D antibodies also had anti-Sm-B/B' antibodies. These data indicate that anti-Sm-D antibodies are specific for SLE, whereas anti-Sm-B/B' are not, confirming a previous report (6). The detection of antibodies to Sm-B/B' in the absence of anti-Sm-D antibodies in patients with MCTD is most probably attributable to the reported cross-reactivity of antibodies to RNP-A and RNP-C with Sm-B/B' (7, 8). Both antigens share proline- rich octapeptide epitopes.

In conclusion, (a) we confirmed that a high percentage of patients with anti-RNP antibodies have Raynaud phenomenon; (b) assay systems composed of recombinant antigens may miss rare samples that contain anti-RNP antibodies; (c) anti-Sm-D antibodies are specific for SLE, whereas anti-Sm-B/B' are not; (d) within a group of patients with antibodies to RNP, no associations between antibodies to individual snU^sub 1^-RNP proteins and symptoms and/or organ involvement were found, except for a possible association between decreased lung diffusion and the presence of antibodies to proteins A and C. The latter finding requires confirmation.

The Varelisa Split ANA Profile assays were kindly provided by Sweden Diagnostics (Freiburg, Germany).

References

1. Sharp GC, Irvin WS, LaRoque RL, Velez C, Daly V, Kaiser AD, et al. Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy. J Clin Invest 1971;50:350-9.

2. Benito-Garcia E, Schur PH, Lahita R, and the American College of Rheumatology ad Hoc Committee on Immunologic Testing Guidelines. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-Sm and anti-RNP antibody tests. Arthritis Rheum 2004;6:1030-44.

3. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725.

4. Alarcn-Segovia D, Villareal M. Classification and diagnostic criteria for MCTD. In: Kasukawa R, Sharp GC, eds. Mixed connective tissue disease and antinuclear antibodies. Amsterdam: Elsevier, 1987:33-40.

5. Hoffman IEA, Peene I, Meheus L, Huizinga TWJ, Cebecauer L, Isenberg D, et al. Specific antinuclear antibodies are associated with clinical features in systemic lupus erythematosus. Ann Rheum Dis 2004;63:1155-8.

6. Gaubitz M, Wagmann C, Schotte H, Willeke P, Domschke W. Differentiation of RNP- and Sm antibody subsets in SLE and MCTD patients by a new ELISA using recombinant antigens. Cell Mol Biol 2002;48:317-21.

7. Arbuckle MR, Schilling AR, Harley JB, James JA. A limited lupus anti-spliceosomal response targets a cross-reactive, proline- rich motif. J Autoimmun 1998;11:431-8.

8. Misaki Y, Yamamoto K, Yanagi K, Miura H, Ichijo H, Kato T, et al. B cell epitope on the U1 snRNP-C autoantigen contains a sequence similar to that of the herpes simplex virus protein. Eur J Immunol 1993;23:1064-71.

Previously published online at DOI: 10.1373/clinchem.2005.053652

Ariane Luyckx,1 Ren Westhovens,2 Els Oris,1 Wolfgang Papisch,3 and Xavier Bossuyt1* (1 Laboratory Medicine and 2 Internal Medicine, University Hospital Leuven, Leuven, Belgium; 3 Sweden Diagnostics, Freiburg, Germany; * address correspondence to this author at: Department of Laboratory Medicine, Immunology, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium; fax 32-13-347042, e- mail xavier.bossuyt@uz.kuleuven.ac.be)

Copyright American Association for Clinical Chemistry Oct 2005

Source: Clinical Chemistry

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