September 1978

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Diagnostic significance of antibody to native deoxyribonucleic acid in children with juvenile rheumatoid arthritis and other connective tissue diseases Sera of children with juvenile rheumatoid arthritis and other connective tissue diseases were tested for antibodies to native DNA by a radio labeled-binding assay. Normal values were obtained in 130 children with IRA, including 28 with uveitis and 14 with selective IgA deficiency. Normal values were also found in sera from children with dermatomyositis, scleroderma, polyarteritis, ankylosing spondylitis, and a variety of other nonconnective tissue diseases. The only sera with elevated DNA-binding assays were from children with systemic lupus erythematosus. On the basis of these data, increased levels of antibodies to native DNA distinguished patients with active SLE from children with JRA.

James T. Cassidy, M.D.,* Sara E. Walker, M.D., Susan J. Soderstrom, M.S., Ross E. Petty, M.D., and Donita B. Sullivan, M.D., Ann

accounts for approximately 75% of children seen in a pediatric rheumatic disease clinic.' The overall prognosis for IRA is relatively good; more than 70% of affected children enter adulthood with normal functional capacity." An important exclusion in the diagnosis of JRA is systemic lupus erythematosus. Early in its course and for a number of years after onset, SLE may be indistinguishable clinically from JRA.a It carries a relatively poor prognosis in children, related in large part to the frequency of development of nephritis.' The importance of laboratory tests which would distinguish between these two diseases is JUVENILE RHEUMATOID AR.THRITIS

Hom the Division of Pediatric Rheumatology and Immunology, Department of Pediatrics and Communicable Diseases, and the Rackham Arthritis Research Unit, Department of Internal Medicine, The University of Michigan Medical School. Supported in part by grants from the Michigan Chapter of The Arthritis Foundation, and by Grant CA 13297 from the National Cancer Institute, The National Institutes oj Health, United States Public Health Service. •Reprint address: R4633 Kresge Medical Research Building, Rackham Arthritls Research Unit, The University 0/ Michigan Medical School, Ann Arbor, M148109.

Vol. 93, No.3, pp. 416·420

Arbor,

Mich.

obvious. Antibody to double-stranded or native deoxyribonucleic acid is a hallmark of active immune-complex disease in the patient with SLE.5 Although early reports of the finding of anti-ds-DNA antibody in children with Abbreviations used JRA: juvenile rheumatoid arthritis SLE: systemic lupus erythematosus DNA: deoxyribonucleic acid RNA: ribonucleic acid ANA: anti-nuclear antibody ds-DNA: double-stranded DNA cpm: counts per minute

JRA have been published,': • it has been our impression

that this test, if properly controlled, is not positive in children with IRA. The purpose of the present study was to compare the results ofa binding assay for anti-ds-DNA antibodies in a large number of sera from children with JRA, SLE, other connective tissue diseases, and control groups. MATERIALS AND METHODS

Study groups. The Table describes the groups of children on whom DNA-binding assays were performed. The

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1978 The C. V. Mosby Co.

DNA in juvenile rheumatoid arthritis

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Table. Groups of children on whom DNA-binding assays were performed Diagnostic category

Juvenile rheumatoid arthritis Children with JRA Children with uveitis and arthritis Children with selective IgA deficiency and arthritis Systemic lupus erythematosus Children with SLE Children with selective IgA deficiency Children who converted from JRA to SLE Dermatomyositis Progressive systemic sclerosis Polyarteritis Juvenile ankylosmg spondylitis Hospitalized children Normal children

Sex

Mean age (yr)

No. of children

E/M

130 28 14

9/41 22/6 12/2

8.8 7.5 9.6

1-17 3-18 3-15

2.8 2.6 3.3

1.5 1.6 1.4

0.1-9.6 0.2-12 1.7-5.7

35 4 8 34 13 12 11 63 60

2817 3/1 6/2 2717 12/1 3/9 1110 34/30 30/30

13.9 15 12.9 10.2 12.1 11.7 15.8 10 11

4-18 12-18 7-18 3-18 7-18 4-18 9-18 1-16 4-18

29 28 25 2.5 4.5 2.9 1.2 1.7 2.4

31 42 31 1.5 2.6 2.2 0.5 1.1 1.4

0.3-94 0.3-90 2.0-97 0.9·7.4 0.9-10 0.8-8.1 0.5-1.8 0.3-4.6 0.7-9.2

first available, serum on each patient was used in this study. All sera had been stored at -70°C or were tested shortly after obtaining from the patient. Blood was obtained by venipuncture, allowed to clot for one hour at room temperature, and centrifuged to separate the serum. All children with connective tissue diseases met commonly accepted criteria for JRA,' SLE,' dermatomyositis." scleroderma," polyarteritis," and juvenile ankylosing spondylitis. " In addition to the 130children with JRA, there were 28 children with JRA and chronic uveitis, and 14 children with JRA and selective IgA deficiency, defined as undectable serum and salivary IgA « 0.01 mg/ml). Active disease was present in 9I% of the children with JRA. The percent of children and mean age of onset for each type of onset were as follows: polyarthritis 46%-6 years, oligoarthritis 360/0-4 years, and systemic onset 18%-6 years. Mean duration of disease for each type of onset at the time of study was, respectively, 3.5, 2.9, and 1.7 years. Thirty-five children with SLE, four with selective IgA deficiency and SLE, and eight children who had been diagnosed originally as having JRA, but who later developed overt SLE, were examined. Sera from normal children were obtained from a community health survey" and consisted of two girls and two boys for each year of age from 4 to 18. Sera were also obtained from 63 hospitalized children with nonconnective tissue diseases. A second serum specimen dated one year after the first, was selected for sequential studies on 28 children with JRA. Eight additional specimens in the group with uveitis were examined a second time. Extraction and characterization of "C-DNA. KB cells"

Age range (yr)

Mean DNAbinding

SD

Range

were grown in spinner culture (200,000 cells/ml) in the presence of 250 /LCi of thymidine-z-vC (54 Ci/rnmole) at 37°C for 72 hours. The labeled DNA was extracted with phenol.> The 2601230 nm spectrophotometric ratio was 2.1, and the 2601280 nm ratio was 1.9,1" The specific activity of the product was 0.06 p,Ci/p,g, assuming an optical density of 1.0 at 260 nm for a solution containing 50 JLg DNA/ml. Protein was not detected by the Lowry test." Ribonucleic acid contamination was not detected by ribonuclease A digestion (Worthington Biochemical Corp.), and NaOH hydrolysis." When the HC-labeled DNA used in the DNA-binding assay was incubated with single-strand specific 8, nuclease, less than 5% of this radioactive substrate was digested. 1 s, 19 DNA-binding assay for anti-DNA antibodies. DNAbinding was determined by a modification of the ammonium sulfate precipitation technique!' Optimal concentrations of test serum and DNA were determined initially on the basis of titration curves constructed from increasing volumesof high-titer SLE serum (2 to 50 J.ll) or HC-Iabeled DNA. The final concentrations chosen represented those that precipitated 95% of the labeled DNA by a high-titer serum from a patient with untreated active lupus nephritis. This serum and a negative control serum were included with each assay. Duplicate determinations of each test serum were averaged. As finally constituted, 15 JLl. of serum were added to 7S JLI of O,l5M borate buffer, pH 8.0, at 4°C in plastic microcentrifuge tubes. Ten microliters of HC-Iabeled DNA (0.034 Jlg, 400 cpm) were employed in each test. The tubes were incubated at 37°C for 30 minutes and then at 4°C for 18 hours. An equal volume of cold saturated ammonium sulfate solution was added to each tube, mixed, and allowed to stand at 4°C

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for 1 hour. The tubes are centrifuged in a Beckman Microfuge for 5 minutes (10,000 X g), and the supernatant fluid was removed. Each pellet was washed with 200 J,U of cold 50% saturated ammonium sulfate solution. After recentrifugation and aspiration of the supernatant, each pellet was dissolved in 200 pl of distilled water. The original supernatants and washes and dissolved pellets were applied separately to one-inch squares of Whatman 3-MMfilter paper. Each dried paper was counted in 20 ml of toluene scintillation fluid for 10 minutes in a Packard Liquid Scintillation Counter. Percent binding was calculated according to the formula: precipitate cpm

100 X precipitate cpm

+ supernatant cprn +

samples of serum from the patients with uveitis showed no significant difference in binding. SLE sera. DNA-binding above 12% was confined to sera of patients with diagnosed SLE. Binding greater than 35%in sera from patients with SLE was not decreased by washing the precipitate. Repeated attempts to demonstrate a heat-labile binding phenomenon in these sera failed. Children with selective IgA deficiency who also had active SLE had elevated DNA-binding. Sera from children with active SLE who had previously been diagnosed as having IRA had increased DNA-binding. Other study groups. Normal subjects, children with other connective tissue diseases, and hospitalized children had binding values of 10% or less.

wash cpm

Effects of heating sera and washing precipitates on percent DNA bound. Eight sera from patients with SLE that demonstrated binding in the 30 to 95% range, and a corresponding number of sera from each of the other control groups, were tested for heat-labile nonspecific binding." No difference was demonstrated after incubation at 56°C for 15, 30, 45, or 60 minutes. Experiments with 32 sera indicated that approximately 20% of the radioactive label could be nonspecifically trapped in the unwashed pellet. This percentage was reduced for normal sera to the range of 1 to 3% binding by a single wash with cold 50% saturated ammonium sulfate solution. Second and third washes did not reduce residual counts further; therefore a single wash was used in this study. DNA-binding of 35% or more was not altered by these manipulations. RESULTS Variability of the method. The replicate error of the method was low: the coefficients of variation for the 95% confidence limits for 94 determinations during the entire course of this study were ± 70% for the negative control serum and ± 5.1% for the SLE control serum. For the negative control serum the mean DNA-binding was 2.1% with a SD of ± 0.9. The respective values for the positive SLE control serum were 94% ± 2.9. JRA sera. No patient with IRA was found who had DNA-binding greater than 9.6%. Patients with IRA and uveitis likewise had low DNA-binding assays (highest 11.7%), as did children with selective IgA deficiency and IRA. Two sequential sera were tested on 28 children with IRA. The means ± 1 SD were: group 1, 2.5 ± 1.2; and group 2, 1.8 ± 0.9. Considering the coefficient of variation for measurement of these low percentages and the fact that both samples gave values consistently in the normal range, we believe that there was no meaningful difference between these two sets of bindings. The two

DISCUSSION Most laboratory tests lack diagnostic specificity and are of limited use in assisting in the differentiation between IRA and SLE. Thus neither LE cells,' ANA,2' nor rheumatoid factors" occur exclusively in one or the other disease category. Antibodies to DNA have been described in the sera of patients with connective tissue diseases, but it is becoming apparent that this phenomenon can be quite specific for SLE.· Among the connective tissue diseases, anti-DNA antibodies have been reported in adults with Sjogren syndrome," uveitis," '4 and adult rheumatoid arthritis .•.•• Two technical problems contribute to apparent nonspecificity of tests for anti-DNA antibodies. (1) A proportion of the radiolabeled DNA is nonspecifically included in the precipitate produced by addition of ammonium sulfate. This represents inclusion within the precipitate space of a small amount of supernatant fluid containing unbound labeled DNA. The error introduced by this factor is greatest when the amount of specific binding is low, and smallest when the amount of specific binding is high.'· This technical problem can be monitored by using a volume indicator such as "Na or by washing the precipitate in half-saturated ammonium sulfate, as was done in this study. In addition, the use of high-speed centrifugation (10,000 X g) yields a firmly packed precipitate which minimizes this problem. (2) The work of Samaha and Irvin" has shown that most preparations of "double-stranded" DNA are substantially contaminated with single-stranded antigen or partly single-stranded DNA, e.g., the ends of the double-stranded helix. Antibodies reacting with single-stranded DNA are not specific for SLE and are found in a wide variety of other diseases and in normal serum.w It is important, therefore, to ascertain the extent to which the antigen used in an assay for anti-ds-DNA antibodies is contaminated with singlestranded DNA. As demonstrated by reaction with single-

Volume 93 Number 3

stranded nuclease, the DNA used in the assays in this study contained less than 5% single-stranded DNA.'9 DNA can be purified further, if heavily contaminated, by gradient centrifugation on cesium chloride;" fractionation on kieselguhr,» hydroxyl apatite," or benzoylated naphthoylated DEAE-cellulose,'2 or by filtration through nitrocellulose filters."" No study of DNA-binding has compared a large number of children with IRA, SLE, and other connective tissue diseases, nor has any study included disease control groups and normal children of similar ages. In the study of Hughes et al," two of 18 patients with JRA had increased DNA-binding. These two patients were initially diagnosed as having JRA, but both subsequently developed SLE and nephritis. Bell et al" described five of 162 sera from patients with IRA which had increased DNAbinding in the range of that in patients with SLE. They commented, however, that these children had some of the clinical manifestations of disease which were strongly suggestive of SLE: one had alopecia, two had pleuritis, two had leukopenia, and two had LE cells. Normal DNAbinding assays were found in another study of25 children with IRA and chronic uveitis, compared to 24 children with IRA who did not have uveitis." In our study, only one patient with IRA and uveitis developed moderate DNA-binding activity (24%) two years after the serum included in the present study, which had a normal binding activity, was drawn. On the basis of the present study, a normal DNAbinding assay is an important criterion in differentiating IRA from active SLE in children. Antinuclear antibodies do not make this distinction. Although the various tests for ANA are sensitive laboratory measures of these autoimmune markers, positive results are not specific for SLE. Both IRA and SLE are associated with a high frequency of ANA positivity, virtually 100% in acute SLE and 40% in JRA." In at least one subgroup of patients with IRA, the child with limited joint disease and uveitis, and frequency of ANA positivity is even higher-70 to 80%.",,3' Antibody to ds-DNA is only one type of ANA, and on the basis of this study does not account for the high frequency of ANA in the sera of children with JRA. The children in this report with systemic onset of JRA, whose disease most resembles SLE clinically, had normal levels of DNA-binding activity. This report has demonstrated that children with IRA have normal levels of DNA-binding in serum. It is conceivable that testing of additional numbers of sera would reveal the expected and inevitable exceptions to this observation. Later in disease, certain children with initially diagnosed IRA may develop antibodies to dsDNA, and overt SLE. A few others may develop these

DNA in juvenile rheumatoid arthritis

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antibodies as a manifestation of uveitis. With these exceptions, and on the basis of the large numbers of children tested in the present program, DNA-binding assays remain a valuable and specific diagnostic indicator of active SLE in childhood. REFERENCES 1. Ansell B: Juvenile chronic polyarthritis: Series 3, Arthritis

Rheum 20(Suppl):176, 1977. 2. Calabro II, and Marchesano JM: The early natural history of juvenile rheumatoid arthritis. A lO-yearfollow-up study of 100 cases, Med Clin North Am 52:567, 1968. 3. Ragsdale C, Petty RE, Cassidy IT, et al: The clinical progression of juvenile rheumatoid arthritis to systemic lupus erythematosus (submitted for publication). 4. Cassidy IT, Sullivan DB, Petty RE, et al: Lupus nephritis and encephalopathy: prognosis in 58 children, Arthritis Rheum 2O(Suppl):315, 1977. 5. Hughes GRY. Cohen SA, and Christian CL: Anti-DNA activity in systemic lupus erythematosus, Ann Rheum Dis 30:259, 1971. 6. Bell C, Talal N, and Schur PH: Antibodies to DNA in patients with rheumatoid arthritis and juvenile rheumatoid arthritis, Arthritis Rheum 18:535, 1975. 7. Brewer EJ Ir, Bass J, Baum J, et al: Current proposed revision of IRA criteria, Arthritis Rheum 20(Suppl):195, 1977. 8. Cohen AS, Reynolds WE, Franklin EC, et al: Preliminary criteria for the classification of systemic lupus erythematosus, Bull Rheum Dis 21:643, 1971. 9. Sullivan DB, Cassidy IT, and Petty RE: Dermatomyositis in the pediatric patient, Arthritis Rheum 20:327, 1977. lO. Dabich L, Sullivan DB, and Cassidy IT: Scleroderma in the child, I PEDIATR 85:770, 1974. II. Magilavy DB. Petty RE, Cassidy IT, et al: A syndrome of childhood polyarteritis, I PEDIATR 91:25, 1977. 12. Ladd JR, Cassidy IT, and Martel W: Iuvenile ankylosing spondylitis, Arthritis Rheum 14:579, 1971. 13. Cassidy IT, Nordby OL, and Dodge JH: Biologicvariation of human serum immunoglobulin concentrations: sex-age specific effects, J Chronic Dis 27:507, 1974. 14. Eagle H: Propagation in fluid medium of a human epidermoid carcinoma, strain KB, Proc Soc Exp Biol Med 89:362, 1955. 15. Pincus T, Schur PH, Rose lA, et al: Measurement of serum DNA-binding activity in systemic lupus erythematosus, N Engl J Med 281:701, 1969. 16. Thomas CA Jr, BernsKI, and Kelly TH Jr: Isolation of high molecular weight DNA from bacteria and cell nuclei, in Cantoni GL and Davies DR, editors: Procedures in Nucleic Acid Research, New York, 1966, Harper & Row, Publishers, p 535. 17. Layne E: Spectrophotometric and turbidimetric methods for measuring proteins, Methods En7.ymoI3:447, 1957. 18. Yogt VM: Purification and further properties of singlestrand-specific nuclease from Aspergillus oryzae, Eur J Biochem 33:192. 1973. 19. Wallcer SE, and Bole GO JR: Selective suppression of autoantibody responses in NZB/NZW mice treated with long-term cyclophosphamide, Arthritis Rheum 18:265, 1975.

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20. Thoburn R, Hurvitz AI, and Kunkel HG: A DNA-binding protein in the serum of certain mammalian species, Proc Nail Acad Sci USA 69:3327, 1972. 21. Petty RE, Cassidy JT, and Sullivan DB: Clinical correlates of antinuclear antibodies in juvenile rheumatoid arthritis, J PEDlATR 83:386, 1973. 22. Cassidy JT, and Yalkenburg HA: A five year prospective study of rheumatoid factor tests in juvenile rheumatoid arthritis, Arthritis Rheum 10:83, 1967. 23. Rheins MS, Burns RM, Suie T, et al: Intermittent appearance of anti-DNA in the sera of uveitis patients, Am J Ophthalmol 66:924, 1968. 24. Epstein WV, Tan M, and Easterbrook M: Serum antibody to double-stranded RNA and DNA in patients with idiopathic and secondary uveitis, N Engl J Med 285: 1502, 1971. 25. Grennan DM, Sloane D, Behan A, et al: Clinical significance of antibodies to native DNA as measured by a DNA binding technique in patients with articular features of rheumatoid arthritis, Ann Rheum Dis 36:30, 1977. 26. Farr RS: A quantitative immunochernical measure of the primary interaction between 1* BSA and antibody, J Infect Dis 103:239, 1958. 27. Samaha RJ, and Irvin WS: Deoxyribonucleic acid stranded-

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ness. Partial characterization of the antigenic regions binding antibodies in lupus erythematosus serum, J Clin Invest 56:446, 1975. Agnello Y, Koffler D, and Kunkel HG: Immune complex systems in the nephritis of systemic lupus erythematosus, Kidney Int 3:90, 1973. Flamm WG, Bond HE, and Burr HE: Density-gradient centrifugation of DNA in a fixed-angle rotor. A higher order of resolution, Biochem Biophys Acta 129:310, 1966. Sueoka N, and Cheng T: Fractionation of DNA on methylated albumin column, Methods Enzymol12:562, 1967. Meinke W, Goldstein DA, and Hall MR: Rapid isolation of mouse DNA from cells in tissue culture, Anal Biochem 58:82, 1974. Medof ME, Locker J, Bennett RM, et al: False positive "native-DNA" binding by non-SLE sera, Clin Res 24:332A, 1976. Nygaard AP, and Hall BD: A method for the detection of RNA-DNA complexes, Biochem Biophys Res Comm 12:98, 1963. Schaller JG, Johnson GD, Holborow EJ, et al: The association of antinuclear antibodies with the chronic iridocyclitis of juvenile rheumatoid arthritis (Still's disease), Arthritis Rheum 17:409, 1974.

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Diagnostic significance of antibody to native deoxyribonucleic acid in children with juvenile rheumatoid arthritis and other connective tissue diseases.

September 1978 416 The Journal ofPE D LA T R L C S Diagnostic significance of antibody to native deoxyribonucleic acid in children with juvenile rh...
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