Journal of Immunogenetics (1977) 4,429432.
G m PHENOTYPES I N AUTOIMMUNE THYROID DISEASE N. R. F A R I D , R .M. N E W T O NE, L K EP. N O E LA N D W. H. M A R S H A L L
Faculty of Medicine, Memorial University of Newfoundland, St John’s, Newfoundland, Canada (Received 22 April 1977)
SUMMARY
The Gm phenotype Gm f,b or Gm f,n,b was found in all forty patients with Graves’ disease studied, contrasted with thirty-five out of forty controls and twenty out of thirty-one patients with thyroiditis. The difference between the two groups with autoimmune thyroid disease was significant. These results suggest that thyroid stimulating antibodies may be allotypically restricted. INTRODUCTION Antibodies play an important role in the immunologically induced damage observed in Hashimoto’s thyroiditis (Calder & Irvine, 1974; Doniach, 1975) and in the hyperstimulation of the thyroid gland resulting in the hyperthyroidism of Graves’ disease (Smith & Hall, 1974). Genetic factors are known to be operative in both these conditions (Doniach, 1975). The search for genetic markers in these diseases has recently resulted in the description of an association between Graves’ disease, but not thyroiditis, and HLA (Farid et al., 1976; Grumet et al., 1974). With t h s in mind, we have studied immunoglobulin G (Gm) allotypes in autoimmune thyroid disease. Gm allotypes of the IgG molecules are coded for by the structural genes for the four G sub-classes. Variants for IgG1, 2, and 3 can be easily identified in the whole serum. Population and family studies have shown that genes coding for different subclass markers are closely linked. Such markers are thus usually inherited as a fixed complex or haplotype. (Natvig & Kunkel, 1973). PATIENTS A N D METHODS Sera from forty patients suffering from Graves’ disease, thirty-one from patients with Hashimoto’s thyroiditis and forty healthy blood donors were studied. All patients and controls were adult Caucasians from the Avalon Peninsula of Newfoundland. The criteria for the diagnosis of thyroiditis were the presence of goitre, high titres of thyroid antibodies, Correspondence: Dr N. R. Farid, Thyroid Research Laboratory, Health Sciences Center, St John’s, Newfoundland A I B 3V6 Canada. 429
430
N . R.Farid et al.
a definitely spotty thyroid scan with or without hormonal evidence of hypothyroidism. In the case of Graves’ disease the diagnosis was based on clinical features, the absence of nodularity of the thyroid gland verified by rectilinear scanning and elevated level of thyroid hormones. Immunoglobulin G allotypes were determined by a passive haemagglutination technique (Morel1 el af.,1972). Typing was carried out for the following antigens: z, a, x, f, n, g, b, s. and t. Statistical analyses were made using v2 tests.
RESULTS Table 1 depicts the Gm phenotypes observed in the control group and the two groups of patients suffering from Graves’ disease and Hashimoto’s thyroiditis. Although
TABLEI . Gm phenotypes among controls and patients with autoimmune thyroid disease Phenotype f. b f.n.b
Controls
Grave’s disease
2
4 19 0 0 9
14
z. a. g 2.a.x.g z. a.g.f. n. b 2.a.x.g.f.n.b z. a.g.f. b 2.a.x.g.f. b others’
2 3 7
Total
40
Hashimoto’s thyroiditis
4
6 I 3 2
2 2 0 31
40 ~
~-
~
Various phenotypes were combined into four groups for valid statistical comparisons to be made. Others include the phenotypes z,a.f,n, b and z,a,x,f.n,b among controls and 2.a.x.f. b and z.a.x,g.s,t among patients with thyroiditis. z,a,x,g,s,t is a rare phenotype a m o n g Caucasians.
heterogeneity in IgG allotypes was observed between controls and each of the Graves’ disease = 5.8564) and the thyroiditis groups (x’ = 5.657), statistical significance was not achieved (P > 0.05 for 3 d.f.). However, a significant difference (x’ = 24.492, P < 0.00025) between the two thyroid disease groups was observed. When Grn haplotypes were considered, it was clear that all forty patients with Graves’ disease carried the haplotypes f,n,b or f,b, whereas only thirty-five out of forty controls (x’ with Yates’ correction = 5.5466, P < 0.025) and twenty out of thirty-one patients with thyroiditis did (x’ = 5.2736. P < 0.025). An even greater divergence in the frequency of the haplotypes f,n,b and f,b was observed between the Graves’ disease and thyroiditis groups (x* = 16.5848, P < 0.0005). Conversely, the haplotypes z,a,g and z,a,x,g were increased in the thyroiditis group and decreased in the Graves’ disease group, as compared to controls. The differences. however, were not significant. The difference between the two thyroid disease groups was significant (x2with Yates’ correction = 8.7839, P < 0.005).
Gm phenotypes in autoimmune thyroid disease
43 1
Haplotype frequencies were estimated by the counting method of maximum likelihood. X* tests for goodness of fit gave values of 7.1 for controls, 11.8 for the thyroiditis group and 6.6 for the Graves’ disease group, all of which were not significant (P 2 0.4). This
indicates a good fit between the observed and expected haplotype frequencies in the three groups studied. DISCUSSION Previous studies failed to show an association of common Gm types with diseases (Steinberg, 1962). However, it has been suggested that the phenotype a,g may be associated with improved survival following various illnesses (MacKay et af., 1975). There is also evidence that the haplotype f,n,b is associated with increased levels of the IgG subclasses 2, 3 and 4 (Yount et af., 1976; Kunkel et af., 1970). Concerning specific antibody response, it has been reported that the phenotype a,g was associated with an augmented immune response to flagellin antigen from Salmonella adeliade and f,b with low responses (Wells et al., 1971). Uncommon Gm phenotypes have been found in association with considerably reduced concentrations of IgG subclasses and immunodeficiency (Yount et af., 1970), and more recently with neuroblastoma (Morel1 ef af., 1977). The present investigation reports for the first time an association between Gm allotypes and autoimmune thyroid disease. The divergence of Gm phenotypes in each of the two thyroid disease groups from controls almost certainly represents a genuine difference. Both controls and patients were randomly selected and were demographically similar. Haplotype frequencies among our group of forty controls is identical to that reported for Caucasian populations (Johnson et af., 1977). Thus the frequency of the haplotype f,n,b/f,b is 0.6375 among our control population. Our results suggest that Gm typing is likely to be a useful genetic marker for Graves’ disease, in addition to HLA (Grumet et-al., 1974; Farid ef af., 1976) and phenylthiocarbamide (PTC) tasting (Kitchin ef al., 1959), and is possibly the first marker to be described for thyroiditis. The most obvious implication of our finding of divergence of Gm phenotypes among the two groups of patients with autoimmune thyroid disease is that Graves’ disease and Hashimoto’s thyroiditis are probably two immunogenetically distinct diseases. This has also been previously suggested by HLA data (Farid et af., 1976). The fact that all patients with Graves’ disease carry the haplotype f,n,b/f,b suggests that the autoantibodies directed against the thyroid membrane may be allotypically restricted. Although data are not available as to the homogeneity or otherwise of human thyroid-stimulating antibodies, earlier observations on the long-acting thyroid stimulator (assayed in mouse) strongly suggest that these antibodies are heterogeneous (Kriss, 1968; Adlkofer el af., 1973). The association between specific allotypes and antibody specificity in Graves’ disease is therefore unlikely due to linkage to idiotypic determinants (Weigert et af., 1975). ACKNOWLEDGMENTS
Antisera for Gm typing were kindly provided by Dr Erna van Loghem. We wish to thank Dr Bodil Larsen for helpful advice, Dr David Bryant for statistical advice and Miss Roxanne Butler for typing the manuscript. This work is supported by grants from the Medical Research Council of Canada.
N . R. Farid et al.
432
REFERENCES ADLKOFER, F., SCHLEUSENER, H., UHEE, L., ANANOS,A. & BRAMMIER,C. (1973) Heterogeneity of long
acting thyroid stimulator (LATSF-activity, thyroglobulin antibodies and thyroid microsomal antibodies. Acla Endocrinologica, 13,483.
CALDER,E.A. & IRVINE,W.J. (1975) Cell-mediated immunity and immune complexes in thyroid disease. Clinics in Endocrinology and Metabolism, 4, 287. DOHIACH,D. (1975) Humoral and genetic aspects of thyroid autoimmunity. Clinics in Endocrinology and Metabolism, 4, 267. FARID,N.R., BARNARD, J.M. & MARSHALL,W.H. (1976) The association of HLA with autoimmune thyroid disease in Newfoundland: the influence of HLA homozygozity in Graves’ disease. Tissue Antigens, 8, 181. GRUMET,F.C., PAYNE,R.O.,KONISHI,J. & KRISS, J.P. (1974) HLA antigens as markers for disease susceptibility and autoimmunity in Graves’ disease. Journal of Clinical Endocrinology and Metabolism, 39, 1 115. JOHNSON. W.E.. KOHN,P.H. & STEINBERG, A.G. (1977) Population genetics of human allotypes Gm Imv and A2m-an analytical review. Clinical Immunologv and Immunopathology, 7,97. KTTCHIN, E.D., HOWELL-EVANS, W., CLARKE,C.A., MCCONNELL, R.B. & SHEPPARD, P.M. (1959) PTC taste response and thyroid disease. British Medical Journal, 1, 1069. KRISS,J.P. (1968). Inactivation of long-acting thyroid stimulator (LATS) by anti-kappa and anti-lambda antisera. Journal of Clinical Endocrinology and Metabolism, 28,1440. KUNKEL,H.G.. JOSLIN.F.G., PENN,G.M. & NATVIG,J.B. (1970) Genetics of yGa globulin. A unique relationship to other classes of F globulin. Journal of Experimental Medicine, 132,508. MACKAY,I.R., WELLS,J.V. & FUDENBERG, H.H. (1975) Correlation of Gm allotypes, antibody response and mortality. Clinical Immunology and Immunopathology, 3,408. MORELL,A., S K A V RE., I ~ STEINBERG, A.G., LOGHEM,E. V A N & TERRY, W.D. (1972) Correlations between the concentrations of the four subclasses of IgG and Gm allotypes in normal human sera. Journal of Immunology, 108. 195. MORELL,A,, KASER,H., SCHERZ,R. & SKAVRJL,S. (1977) Evidence for an association between uncommon Gm phenotypes and neuroblastoma. Lancet, i23. NATVIG, J.B. & KUNKEL.H.G. (1973) Human immunoglobulins: classes, subclasses, genetic variants and idiotypes. Adoances in Immunology,16, 1. SMITH,B.R. & HALL,R. (1974) Thyroid-stimulating immunoglobulins of Graves’ disease. Lancet, ii, 427. STEINBERG. A.G. (1962) Progress in the study of genetically determined human gamma globulin types (the Gm and Imv groups). Progress in Medical Genetics, 2, 1. WEIGERT.M.. POTTER, M. & SACHS, I). (1975) Genetics of the immunoglobulin variable region. Iinmunogenetics. 1. 5 1 1. WELLS.J.V., FUDENBERC, H.H. & MACKAY, I.R. (1971) Relation of the human antibody response to flagellin to Gm genotypes. Journal of Immunology, 107, 1505. YOUNT. W.J.. KUNKEL.H.G. & LITWIN,S.D. (1967) Studies of the Vi (y2c) subgroup of y-globulin. A relationship between concentration and genetic type among normal individuals. Journal of Experimental Medicine. 125. 177. YOUNT, W.J.. HONG,R., SELIGMAN, M., GOOD, R.A. & KUNKEL, H.G. (1970) Imbalances of gamma globulin subgroups and gene defects in patients with primary hypogammaglobulinemia. Journal of Clinical I n ivstigarion 49, I 9 5 7.
.
G m PHENOTYPES I N AUTOIMMUNE THYROID DISEASE N. R. F A R I D , R .M. N E W T O NE, L K EP. N O E LA N D W. H. M A R S H A L L
Faculty of Medicine, Memorial University of Newfoundland, St John’s, Newfoundland, Canada (Received 22 April 1977)
SUMMARY
The Gm phenotype Gm f,b or Gm f,n,b was found in all forty patients with Graves’ disease studied, contrasted with thirty-five out of forty controls and twenty out of thirty-one patients with thyroiditis. The difference between the two groups with autoimmune thyroid disease was significant. These results suggest that thyroid stimulating antibodies may be allotypically restricted. INTRODUCTION Antibodies play an important role in the immunologically induced damage observed in Hashimoto’s thyroiditis (Calder & Irvine, 1974; Doniach, 1975) and in the hyperstimulation of the thyroid gland resulting in the hyperthyroidism of Graves’ disease (Smith & Hall, 1974). Genetic factors are known to be operative in both these conditions (Doniach, 1975). The search for genetic markers in these diseases has recently resulted in the description of an association between Graves’ disease, but not thyroiditis, and HLA (Farid et al., 1976; Grumet et al., 1974). With t h s in mind, we have studied immunoglobulin G (Gm) allotypes in autoimmune thyroid disease. Gm allotypes of the IgG molecules are coded for by the structural genes for the four G sub-classes. Variants for IgG1, 2, and 3 can be easily identified in the whole serum. Population and family studies have shown that genes coding for different subclass markers are closely linked. Such markers are thus usually inherited as a fixed complex or haplotype. (Natvig & Kunkel, 1973). PATIENTS A N D METHODS Sera from forty patients suffering from Graves’ disease, thirty-one from patients with Hashimoto’s thyroiditis and forty healthy blood donors were studied. All patients and controls were adult Caucasians from the Avalon Peninsula of Newfoundland. The criteria for the diagnosis of thyroiditis were the presence of goitre, high titres of thyroid antibodies, Correspondence: Dr N. R. Farid, Thyroid Research Laboratory, Health Sciences Center, St John’s, Newfoundland A I B 3V6 Canada. 429
430
N . R.Farid et al.
a definitely spotty thyroid scan with or without hormonal evidence of hypothyroidism. In the case of Graves’ disease the diagnosis was based on clinical features, the absence of nodularity of the thyroid gland verified by rectilinear scanning and elevated level of thyroid hormones. Immunoglobulin G allotypes were determined by a passive haemagglutination technique (Morel1 el af.,1972). Typing was carried out for the following antigens: z, a, x, f, n, g, b, s. and t. Statistical analyses were made using v2 tests.
RESULTS Table 1 depicts the Gm phenotypes observed in the control group and the two groups of patients suffering from Graves’ disease and Hashimoto’s thyroiditis. Although
TABLEI . Gm phenotypes among controls and patients with autoimmune thyroid disease Phenotype f. b f.n.b
Controls
Grave’s disease
2
4 19 0 0 9
14
z. a. g 2.a.x.g z. a.g.f. n. b 2.a.x.g.f.n.b z. a.g.f. b 2.a.x.g.f. b others’
2 3 7
Total
40
Hashimoto’s thyroiditis
4
6 I 3 2
2 2 0 31
40 ~
~-
~
Various phenotypes were combined into four groups for valid statistical comparisons to be made. Others include the phenotypes z,a.f,n, b and z,a,x,f.n,b among controls and 2.a.x.f. b and z.a.x,g.s,t among patients with thyroiditis. z,a,x,g,s,t is a rare phenotype a m o n g Caucasians.
heterogeneity in IgG allotypes was observed between controls and each of the Graves’ disease = 5.8564) and the thyroiditis groups (x’ = 5.657), statistical significance was not achieved (P > 0.05 for 3 d.f.). However, a significant difference (x’ = 24.492, P < 0.00025) between the two thyroid disease groups was observed. When Grn haplotypes were considered, it was clear that all forty patients with Graves’ disease carried the haplotypes f,n,b or f,b, whereas only thirty-five out of forty controls (x’ with Yates’ correction = 5.5466, P < 0.025) and twenty out of thirty-one patients with thyroiditis did (x’ = 5.2736. P < 0.025). An even greater divergence in the frequency of the haplotypes f,n,b and f,b was observed between the Graves’ disease and thyroiditis groups (x* = 16.5848, P < 0.0005). Conversely, the haplotypes z,a,g and z,a,x,g were increased in the thyroiditis group and decreased in the Graves’ disease group, as compared to controls. The differences. however, were not significant. The difference between the two thyroid disease groups was significant (x2with Yates’ correction = 8.7839, P < 0.005).
Gm phenotypes in autoimmune thyroid disease
43 1
Haplotype frequencies were estimated by the counting method of maximum likelihood. X* tests for goodness of fit gave values of 7.1 for controls, 11.8 for the thyroiditis group and 6.6 for the Graves’ disease group, all of which were not significant (P 2 0.4). This
indicates a good fit between the observed and expected haplotype frequencies in the three groups studied. DISCUSSION Previous studies failed to show an association of common Gm types with diseases (Steinberg, 1962). However, it has been suggested that the phenotype a,g may be associated with improved survival following various illnesses (MacKay et af., 1975). There is also evidence that the haplotype f,n,b is associated with increased levels of the IgG subclasses 2, 3 and 4 (Yount et af., 1976; Kunkel et af., 1970). Concerning specific antibody response, it has been reported that the phenotype a,g was associated with an augmented immune response to flagellin antigen from Salmonella adeliade and f,b with low responses (Wells et al., 1971). Uncommon Gm phenotypes have been found in association with considerably reduced concentrations of IgG subclasses and immunodeficiency (Yount et af., 1970), and more recently with neuroblastoma (Morel1 ef af., 1977). The present investigation reports for the first time an association between Gm allotypes and autoimmune thyroid disease. The divergence of Gm phenotypes in each of the two thyroid disease groups from controls almost certainly represents a genuine difference. Both controls and patients were randomly selected and were demographically similar. Haplotype frequencies among our group of forty controls is identical to that reported for Caucasian populations (Johnson et af., 1977). Thus the frequency of the haplotype f,n,b/f,b is 0.6375 among our control population. Our results suggest that Gm typing is likely to be a useful genetic marker for Graves’ disease, in addition to HLA (Grumet et-al., 1974; Farid ef af., 1976) and phenylthiocarbamide (PTC) tasting (Kitchin ef al., 1959), and is possibly the first marker to be described for thyroiditis. The most obvious implication of our finding of divergence of Gm phenotypes among the two groups of patients with autoimmune thyroid disease is that Graves’ disease and Hashimoto’s thyroiditis are probably two immunogenetically distinct diseases. This has also been previously suggested by HLA data (Farid et af., 1976). The fact that all patients with Graves’ disease carry the haplotype f,n,b/f,b suggests that the autoantibodies directed against the thyroid membrane may be allotypically restricted. Although data are not available as to the homogeneity or otherwise of human thyroid-stimulating antibodies, earlier observations on the long-acting thyroid stimulator (assayed in mouse) strongly suggest that these antibodies are heterogeneous (Kriss, 1968; Adlkofer el af., 1973). The association between specific allotypes and antibody specificity in Graves’ disease is therefore unlikely due to linkage to idiotypic determinants (Weigert et af., 1975). ACKNOWLEDGMENTS
Antisera for Gm typing were kindly provided by Dr Erna van Loghem. We wish to thank Dr Bodil Larsen for helpful advice, Dr David Bryant for statistical advice and Miss Roxanne Butler for typing the manuscript. This work is supported by grants from the Medical Research Council of Canada.
N . R. Farid et al.
432
REFERENCES ADLKOFER, F., SCHLEUSENER, H., UHEE, L., ANANOS,A. & BRAMMIER,C. (1973) Heterogeneity of long
acting thyroid stimulator (LATSF-activity, thyroglobulin antibodies and thyroid microsomal antibodies. Acla Endocrinologica, 13,483.
CALDER,E.A. & IRVINE,W.J. (1975) Cell-mediated immunity and immune complexes in thyroid disease. Clinics in Endocrinology and Metabolism, 4, 287. DOHIACH,D. (1975) Humoral and genetic aspects of thyroid autoimmunity. Clinics in Endocrinology and Metabolism, 4, 267. FARID,N.R., BARNARD, J.M. & MARSHALL,W.H. (1976) The association of HLA with autoimmune thyroid disease in Newfoundland: the influence of HLA homozygozity in Graves’ disease. Tissue Antigens, 8, 181. GRUMET,F.C., PAYNE,R.O.,KONISHI,J. & KRISS, J.P. (1974) HLA antigens as markers for disease susceptibility and autoimmunity in Graves’ disease. Journal of Clinical Endocrinology and Metabolism, 39, 1 115. JOHNSON. W.E.. KOHN,P.H. & STEINBERG, A.G. (1977) Population genetics of human allotypes Gm Imv and A2m-an analytical review. Clinical Immunologv and Immunopathology, 7,97. KTTCHIN, E.D., HOWELL-EVANS, W., CLARKE,C.A., MCCONNELL, R.B. & SHEPPARD, P.M. (1959) PTC taste response and thyroid disease. British Medical Journal, 1, 1069. KRISS,J.P. (1968). Inactivation of long-acting thyroid stimulator (LATS) by anti-kappa and anti-lambda antisera. Journal of Clinical Endocrinology and Metabolism, 28,1440. KUNKEL,H.G.. JOSLIN.F.G., PENN,G.M. & NATVIG,J.B. (1970) Genetics of yGa globulin. A unique relationship to other classes of F globulin. Journal of Experimental Medicine, 132,508. MACKAY,I.R., WELLS,J.V. & FUDENBERG, H.H. (1975) Correlation of Gm allotypes, antibody response and mortality. Clinical Immunology and Immunopathology, 3,408. MORELL,A., S K A V RE., I ~ STEINBERG, A.G., LOGHEM,E. V A N & TERRY, W.D. (1972) Correlations between the concentrations of the four subclasses of IgG and Gm allotypes in normal human sera. Journal of Immunology, 108. 195. MORELL,A,, KASER,H., SCHERZ,R. & SKAVRJL,S. (1977) Evidence for an association between uncommon Gm phenotypes and neuroblastoma. Lancet, i23. NATVIG, J.B. & KUNKEL.H.G. (1973) Human immunoglobulins: classes, subclasses, genetic variants and idiotypes. Adoances in Immunology,16, 1. SMITH,B.R. & HALL,R. (1974) Thyroid-stimulating immunoglobulins of Graves’ disease. Lancet, ii, 427. STEINBERG. A.G. (1962) Progress in the study of genetically determined human gamma globulin types (the Gm and Imv groups). Progress in Medical Genetics, 2, 1. WEIGERT.M.. POTTER, M. & SACHS, I). (1975) Genetics of the immunoglobulin variable region. Iinmunogenetics. 1. 5 1 1. WELLS.J.V., FUDENBERC, H.H. & MACKAY, I.R. (1971) Relation of the human antibody response to flagellin to Gm genotypes. Journal of Immunology, 107, 1505. YOUNT. W.J.. KUNKEL.H.G. & LITWIN,S.D. (1967) Studies of the Vi (y2c) subgroup of y-globulin. A relationship between concentration and genetic type among normal individuals. Journal of Experimental Medicine. 125. 177. YOUNT, W.J.. HONG,R., SELIGMAN, M., GOOD, R.A. & KUNKEL, H.G. (1970) Imbalances of gamma globulin subgroups and gene defects in patients with primary hypogammaglobulinemia. Journal of Clinical I n ivstigarion 49, I 9 5 7.
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