0 1992 Harwood Academic Publishers GmbH Printed in,the United States

Autoimmunity, 1992, Vol. 14. pp. 33-36 Reprints available directly from the publisher Photocopying permitted by license only

CIRCULATING ANTIBODIES TO DNA-RELATED ANTIGENS IN PATIENTS WITH AUTOIMMUNE THYROID DISORDERS A. LOVISELLI*, F. VELLUZZI*, R. PALA*, A. MARCELLO*, P. NURCHIS*, A. MATHIEU*, L. BARTALENA**, E. MARTINO** and L. GRASSO** *Istituto di Medicina Inrema, University of Cagliari, Cagliari. Italy, and **lstituto di Endocrinologia. University of Pisa. Pisa. Italy

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(Received March 5 , 1992; in final form August 6,1992)

A high prevalence of antibodies to double-stranded DNA (AbDNAds) has been recently reported in serum of patients with autoimmune thyroid disorders, but the specificity of this finding has been questioned. For this reason, the prevalence of several antibodies to DNA-related nuclear antigens (AbDRENA) has been evaluated in sera of patients with autoimmune and non-autoimmune thyroid disease. The study group included: 46 Graves’ disease patients, 28 Hashimoto’s thyroiditis patients, 25 patients with toxic nodular goitre and I I with non-toxic nodular goitre. Twenty-eight Graves’ patients were retested during methimazole (MMI) therapy, and 5 after radioiodine administration. Twenty-two patients with systemic lupus erythematosus and 28 normal subjects served as positive and negative controls, respectively. AbDRENA included: AbDNAds by RIA or immunofluorescence (IF); antibodies to single-stranded DNA (AbDNAss) and antibodies to histone (AbHist) by ELISA methods; antibodies to nuclear antigens (ANA) by immunofluorescence. RIA values were considered to be abnormal when 2 SD above the mean of normal controls. In our study 13%of Graves’ patients were positive for AbDNAds by RIA: all of them had negative tests by IF; I I% were positive for AbDNAss. 2% for AbHist and 7% for ANA. A comparable prevalence of positive results for AbDNAds by RIA. with negative IF tests, was found in Hashimoto’s thyroiditis patients. No significant changes of antibody levels were observed in Graves’ patients during MMI treatment or after radioiodine administration. A positivity for AbDNAds or AbDNAss was found in 8% of patients with toxic nodular goitre, but in none of those with non-toxic goitre. All patients with systemic lupus erythematosus had positive tests for AbDNAds by both RIA and IF. In conclusion, the results of the present study indicate that: (i) the prevalence of anti-DNA antibodies is low and does not differ in the different groups of thyroid patients, irrespective of the presence of thyroid autoimmunity and/or thyroid hyperfunction; and (ii) different techniques (e.g. RIA vs. IF) may provide discrepant results, thus at least partially explaining differences among various series. KEY WORDS: Anti-DNA antibodies, Graves’ disease, Hashimoto’s thyroiditis, thyroid autoimmunity.

INTRODUCTION

100% with a cut-off value of 10U/ml. Levels of serum AbDNAds above 20 U/ml have been found by the same method in 95% of Graves’ patients and in 28% of Hashimoto’s thyroiditis patients by Murakarni et ai.’, who also reported that antithyroid drug treatment produced a reduction in serum antibody levels, while increased titres were associated with the relapse. of thyrotoxicosis*. Increased serum AbDNAds concentration by RIA have been described also in postpartum thyroid autoimmune disorders by Tachi er aL9 On the other hand, the specificity of AbDNAds values by RIA has been questioned by Baethge et af.“, who failed to detect these antibodies in patients with thyroid autoimmune disorders using immunofluorescence (IF) methods. To address this question, we assayed AbDNAds by both RIA and IF in sera of patients with autoimmune and non-autoimmune thyroid disorders. Antibodies against a panel of DNA-related antigens (AbDRENA) have been determined.

Serum antibodies to double-stranded DNA (AbDNAds) are commonly detected in patients with systemic lupus erythematosus (SLE), especially during the active phase of the disease: radioimmunoassay values above 25 U/ml are found in about 75% of these patients’. High titers of serum AbDNAds by RIA have been occasionally described also in other autoimmune disorders, such as Sjogren’s syndrome2 and chronic active hepatitis3, but the specificity of this finding has been questioned4”. Recently Katakura er d 6have reported the presence of AbDNAds by RIA in 88% of patients with hyperthyroidism due to Graves’ disease when the cutoff value was 20 U/ml; such a prevalence increased in

Correspondence to: Prof Enio Martino, lstituto di Endocrinologia. University of Pisa, Vide del Tirreno, 64,56018 Tirrenia-Pisa, Italy.

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34

PATIENTS AND METHODS

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Patients

A. LOVISELLI ET AL.

controls. Intra- and interassay coefficients of variation were 5.5 and 10.9%, respectively. ANA was assayed by indirect immunofluorescence on Hep-2 cells (Ortho Diagnostics) in all patients with thyroid disorders and in normal controls: antibody concentration was expressed as dilution titres, the starting dilution being 1:40. All IF tests were carried out with a blind procedure. Data were expressed as meanfSD. Results were analyzed by two-tailed Student’s f-test for paired and unpaired data: the level of significance was PcO.05.

Serum AbDRENA was determined in the following subjects: 46 patients with untreated Graves’ disease (41 women, 5 men, age range 10-66yr); 28 patients with Hashimoto’s thyroiditis (all women, 10 euthyroid, I8 hypothyroid, age range 16-79 yr); 25 patients with toxic nodular goitre (19 women, 6 men, age range 28-71 yr); 11 patients with non-toxic nodular goitre (9 women, 2 men, age range 25-56yr); 22 patients with SLE (20 women, 2 men, age range 27-54 yr); 28 healthy subjects (18 women, 10 men, age range 18-45 yr), normal for hormonal and serological thyroid status. AbDNAds levels were RESULTS measured by RIA also after restoration of euthyroidTable 1 summarizes the results of DNA antibody ism by methimazole (MMI) treatment (2-17 months) determinations in the different groups of subjects in 28 patients or after radioiodine administration in 5 enrolled in this study. All SLE patients had positive patients with Graves’ disease. tests for AbDNAds by both RIA and IF, whereas norThe diagnosis of thyroid disorder was based on mal controls had negative tests for all AbDRENA. clinical grounds, on serum concentrations of total and MeanfSD values in normal controls were as follows: free thyroid hormones and TSH, on the presence or AbDNAds by RIA 7.1f3.5 U/ml (range 1-13), absence of thyroid-directed autoantibodies, and on AbDNAss by ELISA 212f92 U/ml (range 7 1-242), thyroid ultrasound and scan imaging. AbHist by ELISA 201f56 U/ml (range 21-312). All All subjects were residents of Sardinia, Italy; none normal subjects had negative IF tests for AbDNAds of them had clinical or biochemical evidence of conand ANA. nective tissue disease. As shown in Table 1, in Graves’ patients positive AbDNAds tests by RIA were found in 6/46 (13%, mean values 8.0f6.7 U/ml, P=NS vs normals). This Methods positivity was not confirmed by IF. Only few patients Serum total and free thyroid hormones were assayed had positive AbDNAss, AbHist or ANA tests (Table by commercial RIAs; serum TSH was measured by 1). Similar results, and in particular the discrepancy in sensitive IRMA. Anti-thyroglobulin, and anti-thyroid the measurement of AbDNAds by RIA and IF, were peroxidase antibodies were measured by passive hern- observed in Hashimoto’s thyroiditis patients (Table 1). With the exception of two positive tests for agglutination and by RIA, respectively. AbDRENA tests included: AbDNAds, AbDNAss, AbDNAds by RIA (and one by IF), all patients with AbHist and antibodies to nuclear antigens (ANA). non-autoimmune thyroid disorders had negative tests AbDNAds was determined by both RIA and IF. The for AbDRENA (Table 1). Treatment of hypothyroidism by either MMI or RIA method (IM 77/77 1 kit, Amersham, Buckinghamshire, UK) was performed in all patients and in con- radioiodine was not associated with the appearance of trols. In this method, radioiodinated DNA is double- positive tests in patients who were negative prior to stranded, thus avoiding cross-reactivity with single- therapy nor with significant changes in the mean stranded DNA; results were expressed as U/ml. Sera AbDNAds concentration. Among the four Graves’ were considered to be positive when providing results patients who had positive tests before MMI treatment, 2 SD above the mean normal values. Intra- and three became negative and one remained positive, interassay coefficients of variations were 4.5 and with no significant variation in antibody titre (data not 5.5%, respectively. The IF method was performed in shown). all patients with thyroid disorders and in all controls; in this method AbDNA binds to the kinetoplast of hemoflagellate “Crithidia Luciliae” which contains DISCUSSION pure double-stranded DNA”. AbDNAss (tested in 98 thyroid patients) and AbHist were measured in Previous studies have reported tha: circulating patients and in normal controls by an ELISA method AbDNAds were detectable in 88-100% of untreated (BioHyTech Ramat Gan, Israel); values were Graves’ patients and in 28% of Hashimoto’s thyroidexpressed as U/ml, the upper normal limit being itis patients’,’ when values above 10-20 U/rnl were defined as the mean +2 SD of values found in normal taken as abnormal. In the present study, with a cut-off

ANTI-DNA ANTIBODIES IN AUTOIMMUNE THYROID DISORDERS

35

Table 1 Prevalence of AbDRENA in patients with autoimmune and non-autoimmune thyroid disorders and in control ErouDs.

IF

AhDNAss ELISA

AhHisr

RIA

AhDNAils

ELISA

ANA IF

+ve (%)

+ve (%)

+\‘e (%)

+ve (%)

+ve(%)

Thyroid autoimmune disorders Graves’ disease Hashimoto’s thyroiditis

6/46 ( 13) 4/28 (14)

0146 OD8

4/36 ( I I ) 4/28 (14)

Thyroid non-autoimmune disorders Toxic nodular goitre Nontoxic nodular goitre

2/25 (8) O/I I

1/25 (4) O/I 1

OD5

0/25

O/I I

O/I I

OD5 O/I I

Normal controls

OD8

OD8

ona

OD8

OD8

SLE

22/22

22/22

NT

NT

NT

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NT-not ieacd.

value of 14.2 U/ml (2 SD above the mean values in serum components other than specific antibodies normal controls), positive tests for AbDNAds have (anti-phospholipid antibodies, complement fractions), been obtained by RIA in 13% of Graves’ patients and therefore suggesting a further reduction of really 14% of Hashimoto’s patients. Interestingly, mean abnormal cases. Another possible explanation for the discrepancy absolute values in Graves’ patients and in normal controls did not differ. Our results confirm the data concerning the prevalence of AbDNAds in the various might reside in the different cut-off values. In . reported by Swaak er al.12and by McDermott ef ~ 1 . ’ ~ series who failed to find a substantial increase in serum this regard, it should, however, be noted that our cutAbDNAds by RIA in patients with autoimmune thy- off value for AbDNAds was not substantially different roid disorders. AbDNAds have been occasionally from that used by other authors who found a high found in autoimmune disorders other than SLE’.’, and prevalence of these antibodies’.’. Even using a Swaak et al.” have suggested that the finding of circu- 10 U/ml cut-off value, the prevalence of AbDNAds in lating AbDNAds in asymptomatic subjects might be a our series would not be greater than 32% in Graves’ predictive index for the subsequent development of disease and 2 1 % in Hashimoto’s thyroiditis. Another cause of discrepancy might be the selection autoimmune disease. On the other hand, Menard ef u I . ~ pointed out that positivity for AbDNAds in dis- of patients, with particular regard to the metabolic eases other than SLE might be artifactual, due to the status. In this respect it is worth noting that our study presence of DNAss in nucleic acid preparation. In the failed to show any substantial change in the prevaassay we have utilized for AbDNAds determination, lence and/or titer of AbDRENA during MMI treatthe labeled DNA consists of pure double-stranded ment or following radioiodine administration for molecules obtained by enzymatic procedure, thus Graves’ hyperthyroidism. This finding, in disagree. limits the role of avoiding any interference by DNAss. As a matter of ment with Murakami et ~ 1 . ~also fact, only one of the six sera positive for AbDNAds by AbDRENA determination in the evaluation of treatRIA also had abnormal levels of AbDNAss. In ment of hyperthyroidism and in the prediction of addition, the possible interference of other AbDRENA recurrences after treatment withdrawal. An additional explanation for discrepant results of in the AbDNAds is not supported by the substantially negative results of AbHist and/or ANA tests found in the different series might be that autoimmune thyroid our ‘series. Taken together, these data emphasize the disorders are genetically different in the various ethconcept that the figures that we and have nic groups. In this regard, we have recently failed to proposed concerning the low prevalence of observe the linkage of Graves’ disease or Hashimoto’s AbDRENA in autoimmune thyroid disorders are real. thyroiditis in Caucasian populations with HLA-B8 Furthermore, the observation that AbDNAds by IF or -DR3 in Sardinian subjects, for whom a close were substantially negative in our series, on one hand association with HLA-DR2 has been reportedI4. How supports the concept that the anti-DNA antibody this may affect the prevalence of AbDRENA is prevalence is very low in patients with autoimmune unclear. thyroid disorders, on the other hand it underscores the From a practical point of view, on the basis of this greater value of quantitative methods, such as RIA, and previous studie~l’-‘~.determination of serum for its detection. The discrepancy between prevalence AbDRENA appears to be of limited value, if any, in of positive test for AbDNAds by IF as compared to patients with autoimmune (and non-autoimmune) thyRIA might be due to nonspecific binding to DNA of roid disorders.

A. LOVISELLI ET AL.

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Acknowledgements We wish to thank Mr G. Salk for his expert technical assistance. This study was supported by the National Research Council (C.N.R.), Rome, Italy, Target Project "Prevention and Control of Disease Factors (FATMA)".

References 1. Holian J, Griffiths ID, Glass DN, Maini RN, Scott TJ. Human

Autoimmunity Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.

antiDNA antibody: reference standard for diagnosis and management of systemic lupus erythematosus. Ann Rheum Dis 1975; 34: 4 3 8 4 4 3 2. Permin H, Halberg P, Christiansen E. Antibodies against double-stranded DNA in patients with connective tissue diseases. Acra Med Scand 1978; 203: 61-65 3. Iain S. Markham SI, Thomas HC. Sherlock S. Double-stranded DNA-binding capacity of serum in acute and chronic liver disease. Clin Exp Immunol 1976; 26: 35-41 4. Menard HA, Raptis L. Absence d'anticorps anti ADN natif au cors de I'hepatite chronique active. Nouv Presse Med 1979; 8: 3973-3974 5. Gayral-ta Minh M. Foumier GI. Conte JJ. Quels anticorps antiADN? Nouv Presse Med 1979; 8: 3974 6. Katakura M, Yamada T, Aizawa T, Hiramatsu K, Yukimura Y, lshihara M, Takasu 0. Maruyama K, Kameko M, Kanai M. Kobayashi I. Presence of antideoxyribonucleic acid antibody in

patients with hyperthyroidism of Graves' disease. J Clin Endocrinol Merub 1987; 64: 4 0 5 4 0 8

7. Murakami M, Koizumi Y. Aizawa T, Yamada T, Takahashi Y, Watanabe T, Kamoi K. Studies of thyroid function and immune parameters in patients with hyperthyroid Graves' disease in remission. J Clin Endocrinol Metab 1988; 66: 103-108 . 8. Murakami M, Koizumi Y, Aizwa T, Yamada T, Ishihara M, Haseegawa Y, Ogata H, Kamoi K. Kaneko K. Improvement of immunological abnormalities associated with hyperthyroidism of Graves' disease during methimazole treatment. Hurm Metabol Res 1988; 20: 235-238 9. Tachi J, Amino N. Iwatani Y. Tamaki H, Mori M, Aozasa M. Tanizawa 0, Miyai K. Increase in antideoxyribonucleic acid

antibody titer in postpartum aggravation of autoimmune thyroid disease.J Clin Endocrinol Merub 1988; 67: 1049-1053 10. Baethge BA, Levine SN, Wolf RE. Antibodies to nuclear antigens in Graves' disease. J Clin Endocrinol Merub 1988; 66: 485488 I I . Crowe W, Kushner I. An immunofluorescent method using Crithidia Luciliae to detect antibodies to double-stranded DNA. Arthritis Rheum 1977; 20: 8 1 1-8 14 12. Swaak AJG. Groenwold I, Aarden LA, Feltkamp TEW. Detection of anti-ds DNA as diagnostic tool. Rheum Dis 1981; 40: 4549 13. McDermott MT, West SG,Elmen IW,Kidd GS. Antideoxyribonucleic acid antibodies in Graves' disease. J Clin Endocrino1 Merub 1990 7: 509-5 1 1 14. La Nasa G, Carcassi C. Martino E, Mulargia M. Velluzzi F, Vacca A, Grasso L, Loviselli A. Contu L. HLA e tireopatie autoimmuni in Sardegna. Vlll Giornare lralione della Tiroide. Cagliari. 5-7 Dicembre 1990; 63 (Abstract)

Circulating antibodies to DNA-related antigens in patients with autoimmune thyroid disorders.

A high prevalence of antibodies to double-stranded DNA (AbDNAds) has been recently reported in serum of patients with autoimmune thyroid disorders, bu...
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