0021-972X/90/7105-1131$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
Susceptibility to Thyroid Autoimmune Disease: Molecular Analysis of HLA-D Region Genes Identifies New Markers for Goitrous Hashimoto's Thyroiditis K. BADENHOOP*, G. SCHWARZ, P. G. WALFISHf, V. DRUMMOND, K. H. USADEL, AND G. F. BOTTAZZO //. Med. Klinik, Klinikum Mannheim, Universitdt Heidelberg, Federal Republic of Germany; Department of Diabetes and Immunogenetics, St. Bartholomew's Hospital, and Department of Immunology, University College and Middlesex School of Medicine, London, United Kingdom, and Department of Medicine, Endocrine Division and Samuel Lunenfeld Research Institute of Mount Sinai Hospital, University of Toronto School of Medicine, Toronto, Ontario, Canada
ABSTRACT. Hashimoto's thyroiditis has been shown to be associated with the HLA-specificities DR4 and DR5. Since former association studies yielded variable results, we used novel molecular typing methods to assess predisposing immunogenetic factors. Gene analysis of the HLA-DR-DQ and tumor necrosis factor region was performed in a group of Hashimoto's thyroiditis patients and randomly chosen controls using standards and nomenclature of the 10th International Histocompatibility Workshop. Genomic DNA of patients and controls was analyzed using a cDNA probe of the DQB1 gene. The resulting restriction fragment patterns allowed the determination of newly defined DQw-types 1-9. We find the strongest relative risk conferred by DQw7 (RR = 4.7), that is observed in 36 of 64 patients (56%) and only 21 of 98 controls (21%) (P corr < 0.002). Comparison
H
ASHIMOTO'S thyroiditis is an autoimmune disease of the thyroid, characterized by autoantibodies to thyroglobulin and thyroperoxidase (thyroid microsomal antigen) (1). Its prevalence varies in different countries and it can be found in several members of a family or in patients with a family history of thyroid disease (2). Genetic factors predispose to this disease, like other autoimmune diseases, as shown by markers of the HLA region. The HLA region is on the short arm of chromosome 6, spanning from the class II region (HLA DP, DQ, DR) over the class III region (complement, 21-hydroxReceived April 2, 1990. Address requests for reprints to: Dr. K. Badenhoop, Klinikum Mannheim der Universitat Heidelberg, Theodor Kutzer Ufer, D 6800 Mannheim 1, Federal Republic of Germany. *Recipient of a grant from the German Academic Exchange Service and supported by the Deutsche Forschungsgemeinschaft (Ba 976/2-1), the Deutsche Gesellschaft fur Innere Medizin and Boehringer Mannheim Research Fund. fSupported in part by grants from the Physicians of Ontario through the Physician Services Incorporated Foundation and the Mount Sinai Hospital Department of Medicine Research Fund.
of DNA sequence variation in the DQBl gene, that is found predominantly in Hashimoto's thyroiditis patients, indicates that codons 45 and 57 are critical features in DQw7 which distinguish it from other DQw specificities. The adjacent DQA1 genes also display a significant association with Hashimoto's thyroiditis (DQAl*0201/*0301 heterozygotes were found in 37% of patients and 15% controls, P < 0.03). No significant association could be found with polymorphisms of the tumor necrosis factor gene. These results provide a new basis for the concept of genetic susceptibility in Hashimoto's thyroiditis and will help to elucidate the underlying autoimmune mechanisms that lead to disease at the functional level. (J Clin Endocrinol Metab 7 1 : 1131-1137, 1990)
ylase) to the class I region (HLA B, C, A). HLA molecules play a key role in the immune response and antigen recognition by T cells. Hashimoto's thyroiditis has been found to be associated with HLA specificities DR4 and DR5 (3, 4), whereas the atrophic variant, primary myxedema, is associated with HLA-DR3 (for review see Ref. 4). These original studies were based upon HLA serology of the early International Histocompatibility Workshop definitions. Now DNA probes of the DR and DQ genes can be used in a molecular approach to type for DR and DQ specificities (5). This methodology has been standardized in the 10th International Histocompatibility Workshop. It is based upon RFLP of a given DNA probe/ restriction enzyme combination. RFLPs identify patterns of restriction fragments that have been shown to correlate well with serologically and cellularly defined specificities, and in addition detect new specificities. RFLP can therefore be used to type individuals accurately for Class II antigens (6). These fragments also correspond to nucleic acid sequence variation, since sequence analyses of a large number of HLA-DR and -DQ 1131
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BADENHOOP ET AL.
1132
alleles have been published (7). DNA sequence variations can be compared in those critical sequence regions that occur more often in disease associated haplotypes than in controls, as shown in similar studies for type I diabetes mellitus (8). We specifically analyzed the DQB1 gene in Hashimoto's thyroiditis, since both associated specificities DR4 and DR5 are in linkage with DQw3, a specificity encoded by the DQB1 and DQA1 gene. Since DQw3 can be subdivided with the DQB1 RFLP into DQw7 (previously called DQw3.1) and DQw8 (previously DQw3.2), it was of interest, which split would associate with Hashimoto's thyroiditis. Here it is demonstrated for the first time that both DR4 and DR5 are linked to DQw7 in Hashimoto's thyroiditis. DQA1 gene analysis also supports the notion that the DQ region is important for disease susceptibility. .
Subjects and Methods Patients Sixty six Caucasian patients with Hashimoto's thyroiditis were studied; 18 patients were from Toronto, 48 patients from London. The diagnosis of Hashimoto's thyroiditis was based on a palpable goiter, the presence of antimicrosomal antibodies in a titer greater than 1:80 and hypothyroidism documented by decreased total and free T4 and elevated TSH or hyperresponsiveness of serum TSH to TRH stimulation. Controls Ninety eight randomly chosen Caucasian control subjects from London were studied. Careful history taking and family history excluded thyroid or endocrine autoimmune disease. HLA-DR specificities did not differ between a control panel in Toronto and the London group (19). Twenty additional DR4+ controls were kindly provided by Dr. Mytilineos, University of Heidelberg, and four by Dr. Trowsdale, ICRF (London, U.K.). HLA-serology HLA typing for HLA-A, -B, and -C antigens was performed by the standard NIH microlymphocytotoxicity test (9), with selected antisera recognizing ninth Histocompatibility Workshop specificities. HLA-DR, DRw52/53 and DQw typings were performed by double-color fluorescence technique (10). RFLP studies Restriction fragment length polymorphism (RFLP) analysis was performed according to standard procedures recommended by the 10th International Histocompatibility Workshop Southern blotting protocol (11) with some modifications. In brief, DNA was prepared from peripheral blood, and 10 ng were digested with 20 U restriction endonuclease Taql, BamHl, or Ncol at temperatures and buffer conditions specified by the manufacturers (Bethesda Research Laboratories, Amersham,
JCE & M • 1990 Vol 71 • No 5
U.K.). Digests were electophoresed on a 0.8% agarose gel in TAE-buffer (40 mmol/liter trisacetate, 1 mmol/liter EDTA, pH 8), for 24 h at 45 mA. Molecular weight markers, either Hindlll digested lambda DNA with jF/aelll digested phiX or 10th HLA Workshop Southern blot reference markers were run on each gel. The gels were soaked in 0.15 M HC1 for 10 min, then in 0.4 M NaOH for 30 min. DNA was transferred onto Biotrace membranes using the alkaline method (0.4 M NaOH overnight). Filters were prehybridized overnight at 42 C and hybridized at 42 C for 36 h with a radio-labeled cDNA probe in the following mixture: 50% deionized formamide, 0.1% Denhardt's solution, 5 X SSPE, 1% sodium dodecyl sulfate, 5% dextran sulfate and 200 iig/rcA salmon sperm DNA. The filters were washed twice in 2 X SSPE at room temperature for 5 min, then in 2 x SSPE, 0.5% sodium dodecyl sulfate at 65 C for 15 min, and finally in 0.5 x SSPE at 65 C for 15 min. They were then exposed for 3-10 days with intensifying screens on KodakXAR-5 film at -80 C. Assignments of DQw-types by RFLP was done without knowledge of the DR/DQw serotype. Not all patients and controls could be tested for every combination: The following probes/enzyme combinations were used: DRB1/ Taql, DQAl/TagI (51 patients; 46 controls), DQBl/BamHI (64 patients; 98 controls) and TNF/ATcoI (39 patients; 48 controls). Statistical methods RFLP data on DRB1, DQA1, and DQB1 were correlated with serological data (6, 11). Two by 2 tables were analyzed by X2 (with determination of relative risks) or Fisher's exact test as appropriate. Where indicated P values (P corr) were corrected for the numbers of comparisons made (specificities tested in a given probe/enzyme system, e.g. 10 for DR-specificities, 3 for serological DQw-specificities, and 10 for DQw/DQBl RFLP). P values for DQA1, DQA2, and DR4+ DQw7/8 analysis were not corrected. Gene frequencies were determined by direct gene counting (for DQA2 and TNF RFLP).
Results Serological typing for HLA-A, -B, -C, -DR and -DQ antigens Classical serological typing for HLA-A, -B, and -C antigens in patients and controls did not show a statistical significant difference. HLA A2 and B44 were slightly increased in patients, but this did not reach the level of significance (data not shown). HLA-DR specificities, however, differed significantly between patients and controls: As shown in Table 1, HLA DR5 was found in 23 of 66 (35%) patients, but only in 12 of 98 (12%) controls (x2 = 10.7, P corr < 0.01). HLA DR4 was increased in patients too: 33 of 66 (50%) of patients possessed this antigen in contrast to 25 of 98 controls (26%), (x2 = 9.3, P corr < 0.02). HLA-DR4/5 heterozygotes were found in 9 (14%) patients and only 1 (1%) control (P < 0.03, RR = 15.31). The frequency of DR4, DR5, and DR4/5 heterozygotes were similar in patients from London and Toronto (data not shown). The other
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GENETIC SUSCEPTIBILITY TO HASHIMOTO'S THYROIDITIS TABLE 1. HLA DR and DOW antigen frequencies Controls
Hashimoto (n == 66 %) DR1 DR2 DR3 DR4 DR5 DR6 DR7 DR8 DR9 DR10 DR4/5 DRw52 DRw53 DQwl DQw2 DQw3 TABLE 2.
6 (9%) 11 (17%) 17 (26%) 33 (50%) 23 (35%) 12 (18%) 16 (24%) 2 (3%) 2 (3%) 1 (2%) 9 (14%) 47 (71%) 47 (71%) 27 (41%) 32 (50%) 46 (70%)
(n = 98%) 21 (21%) 26 (27%) 25 (26%) 25 (26%) 12 (12%) 19 (19%) 30 (31%) 4(4%) 2 (2%) 1 (1%) 1 (1%) 52 (53%) 65 (66%) 53 (54%) 49 (50%) 45 (46%)
X2 = 9.3 Pc < 0.03 RR = 2.9 X2 = 10.7 Pc
Vol. 71, No. 5 Printed in U.S.A.
Susceptibility to Thyroid Autoimmune Disease: Molecular Analysis of HLA-D Region Genes Identifies New Markers for Goitrous Hashimoto's Thyroiditis K. BADENHOOP*, G. SCHWARZ, P. G. WALFISHf, V. DRUMMOND, K. H. USADEL, AND G. F. BOTTAZZO //. Med. Klinik, Klinikum Mannheim, Universitdt Heidelberg, Federal Republic of Germany; Department of Diabetes and Immunogenetics, St. Bartholomew's Hospital, and Department of Immunology, University College and Middlesex School of Medicine, London, United Kingdom, and Department of Medicine, Endocrine Division and Samuel Lunenfeld Research Institute of Mount Sinai Hospital, University of Toronto School of Medicine, Toronto, Ontario, Canada
ABSTRACT. Hashimoto's thyroiditis has been shown to be associated with the HLA-specificities DR4 and DR5. Since former association studies yielded variable results, we used novel molecular typing methods to assess predisposing immunogenetic factors. Gene analysis of the HLA-DR-DQ and tumor necrosis factor region was performed in a group of Hashimoto's thyroiditis patients and randomly chosen controls using standards and nomenclature of the 10th International Histocompatibility Workshop. Genomic DNA of patients and controls was analyzed using a cDNA probe of the DQB1 gene. The resulting restriction fragment patterns allowed the determination of newly defined DQw-types 1-9. We find the strongest relative risk conferred by DQw7 (RR = 4.7), that is observed in 36 of 64 patients (56%) and only 21 of 98 controls (21%) (P corr < 0.002). Comparison
H
ASHIMOTO'S thyroiditis is an autoimmune disease of the thyroid, characterized by autoantibodies to thyroglobulin and thyroperoxidase (thyroid microsomal antigen) (1). Its prevalence varies in different countries and it can be found in several members of a family or in patients with a family history of thyroid disease (2). Genetic factors predispose to this disease, like other autoimmune diseases, as shown by markers of the HLA region. The HLA region is on the short arm of chromosome 6, spanning from the class II region (HLA DP, DQ, DR) over the class III region (complement, 21-hydroxReceived April 2, 1990. Address requests for reprints to: Dr. K. Badenhoop, Klinikum Mannheim der Universitat Heidelberg, Theodor Kutzer Ufer, D 6800 Mannheim 1, Federal Republic of Germany. *Recipient of a grant from the German Academic Exchange Service and supported by the Deutsche Forschungsgemeinschaft (Ba 976/2-1), the Deutsche Gesellschaft fur Innere Medizin and Boehringer Mannheim Research Fund. fSupported in part by grants from the Physicians of Ontario through the Physician Services Incorporated Foundation and the Mount Sinai Hospital Department of Medicine Research Fund.
of DNA sequence variation in the DQBl gene, that is found predominantly in Hashimoto's thyroiditis patients, indicates that codons 45 and 57 are critical features in DQw7 which distinguish it from other DQw specificities. The adjacent DQA1 genes also display a significant association with Hashimoto's thyroiditis (DQAl*0201/*0301 heterozygotes were found in 37% of patients and 15% controls, P < 0.03). No significant association could be found with polymorphisms of the tumor necrosis factor gene. These results provide a new basis for the concept of genetic susceptibility in Hashimoto's thyroiditis and will help to elucidate the underlying autoimmune mechanisms that lead to disease at the functional level. (J Clin Endocrinol Metab 7 1 : 1131-1137, 1990)
ylase) to the class I region (HLA B, C, A). HLA molecules play a key role in the immune response and antigen recognition by T cells. Hashimoto's thyroiditis has been found to be associated with HLA specificities DR4 and DR5 (3, 4), whereas the atrophic variant, primary myxedema, is associated with HLA-DR3 (for review see Ref. 4). These original studies were based upon HLA serology of the early International Histocompatibility Workshop definitions. Now DNA probes of the DR and DQ genes can be used in a molecular approach to type for DR and DQ specificities (5). This methodology has been standardized in the 10th International Histocompatibility Workshop. It is based upon RFLP of a given DNA probe/ restriction enzyme combination. RFLPs identify patterns of restriction fragments that have been shown to correlate well with serologically and cellularly defined specificities, and in addition detect new specificities. RFLP can therefore be used to type individuals accurately for Class II antigens (6). These fragments also correspond to nucleic acid sequence variation, since sequence analyses of a large number of HLA-DR and -DQ 1131
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 09 November 2015. at 13:00 For personal use only. No other uses without permission. . All rights reserved.
BADENHOOP ET AL.
1132
alleles have been published (7). DNA sequence variations can be compared in those critical sequence regions that occur more often in disease associated haplotypes than in controls, as shown in similar studies for type I diabetes mellitus (8). We specifically analyzed the DQB1 gene in Hashimoto's thyroiditis, since both associated specificities DR4 and DR5 are in linkage with DQw3, a specificity encoded by the DQB1 and DQA1 gene. Since DQw3 can be subdivided with the DQB1 RFLP into DQw7 (previously called DQw3.1) and DQw8 (previously DQw3.2), it was of interest, which split would associate with Hashimoto's thyroiditis. Here it is demonstrated for the first time that both DR4 and DR5 are linked to DQw7 in Hashimoto's thyroiditis. DQA1 gene analysis also supports the notion that the DQ region is important for disease susceptibility. .
Subjects and Methods Patients Sixty six Caucasian patients with Hashimoto's thyroiditis were studied; 18 patients were from Toronto, 48 patients from London. The diagnosis of Hashimoto's thyroiditis was based on a palpable goiter, the presence of antimicrosomal antibodies in a titer greater than 1:80 and hypothyroidism documented by decreased total and free T4 and elevated TSH or hyperresponsiveness of serum TSH to TRH stimulation. Controls Ninety eight randomly chosen Caucasian control subjects from London were studied. Careful history taking and family history excluded thyroid or endocrine autoimmune disease. HLA-DR specificities did not differ between a control panel in Toronto and the London group (19). Twenty additional DR4+ controls were kindly provided by Dr. Mytilineos, University of Heidelberg, and four by Dr. Trowsdale, ICRF (London, U.K.). HLA-serology HLA typing for HLA-A, -B, and -C antigens was performed by the standard NIH microlymphocytotoxicity test (9), with selected antisera recognizing ninth Histocompatibility Workshop specificities. HLA-DR, DRw52/53 and DQw typings were performed by double-color fluorescence technique (10). RFLP studies Restriction fragment length polymorphism (RFLP) analysis was performed according to standard procedures recommended by the 10th International Histocompatibility Workshop Southern blotting protocol (11) with some modifications. In brief, DNA was prepared from peripheral blood, and 10 ng were digested with 20 U restriction endonuclease Taql, BamHl, or Ncol at temperatures and buffer conditions specified by the manufacturers (Bethesda Research Laboratories, Amersham,
JCE & M • 1990 Vol 71 • No 5
U.K.). Digests were electophoresed on a 0.8% agarose gel in TAE-buffer (40 mmol/liter trisacetate, 1 mmol/liter EDTA, pH 8), for 24 h at 45 mA. Molecular weight markers, either Hindlll digested lambda DNA with jF/aelll digested phiX or 10th HLA Workshop Southern blot reference markers were run on each gel. The gels were soaked in 0.15 M HC1 for 10 min, then in 0.4 M NaOH for 30 min. DNA was transferred onto Biotrace membranes using the alkaline method (0.4 M NaOH overnight). Filters were prehybridized overnight at 42 C and hybridized at 42 C for 36 h with a radio-labeled cDNA probe in the following mixture: 50% deionized formamide, 0.1% Denhardt's solution, 5 X SSPE, 1% sodium dodecyl sulfate, 5% dextran sulfate and 200 iig/rcA salmon sperm DNA. The filters were washed twice in 2 X SSPE at room temperature for 5 min, then in 2 x SSPE, 0.5% sodium dodecyl sulfate at 65 C for 15 min, and finally in 0.5 x SSPE at 65 C for 15 min. They were then exposed for 3-10 days with intensifying screens on KodakXAR-5 film at -80 C. Assignments of DQw-types by RFLP was done without knowledge of the DR/DQw serotype. Not all patients and controls could be tested for every combination: The following probes/enzyme combinations were used: DRB1/ Taql, DQAl/TagI (51 patients; 46 controls), DQBl/BamHI (64 patients; 98 controls) and TNF/ATcoI (39 patients; 48 controls). Statistical methods RFLP data on DRB1, DQA1, and DQB1 were correlated with serological data (6, 11). Two by 2 tables were analyzed by X2 (with determination of relative risks) or Fisher's exact test as appropriate. Where indicated P values (P corr) were corrected for the numbers of comparisons made (specificities tested in a given probe/enzyme system, e.g. 10 for DR-specificities, 3 for serological DQw-specificities, and 10 for DQw/DQBl RFLP). P values for DQA1, DQA2, and DR4+ DQw7/8 analysis were not corrected. Gene frequencies were determined by direct gene counting (for DQA2 and TNF RFLP).
Results Serological typing for HLA-A, -B, -C, -DR and -DQ antigens Classical serological typing for HLA-A, -B, and -C antigens in patients and controls did not show a statistical significant difference. HLA A2 and B44 were slightly increased in patients, but this did not reach the level of significance (data not shown). HLA-DR specificities, however, differed significantly between patients and controls: As shown in Table 1, HLA DR5 was found in 23 of 66 (35%) patients, but only in 12 of 98 (12%) controls (x2 = 10.7, P corr < 0.01). HLA DR4 was increased in patients too: 33 of 66 (50%) of patients possessed this antigen in contrast to 25 of 98 controls (26%), (x2 = 9.3, P corr < 0.02). HLA-DR4/5 heterozygotes were found in 9 (14%) patients and only 1 (1%) control (P < 0.03, RR = 15.31). The frequency of DR4, DR5, and DR4/5 heterozygotes were similar in patients from London and Toronto (data not shown). The other
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GENETIC SUSCEPTIBILITY TO HASHIMOTO'S THYROIDITIS TABLE 1. HLA DR and DOW antigen frequencies Controls
Hashimoto (n == 66 %) DR1 DR2 DR3 DR4 DR5 DR6 DR7 DR8 DR9 DR10 DR4/5 DRw52 DRw53 DQwl DQw2 DQw3 TABLE 2.
6 (9%) 11 (17%) 17 (26%) 33 (50%) 23 (35%) 12 (18%) 16 (24%) 2 (3%) 2 (3%) 1 (2%) 9 (14%) 47 (71%) 47 (71%) 27 (41%) 32 (50%) 46 (70%)
(n = 98%) 21 (21%) 26 (27%) 25 (26%) 25 (26%) 12 (12%) 19 (19%) 30 (31%) 4(4%) 2 (2%) 1 (1%) 1 (1%) 52 (53%) 65 (66%) 53 (54%) 49 (50%) 45 (46%)
X2 = 9.3 Pc < 0.03 RR = 2.9 X2 = 10.7 Pc
