0 1992 Harwood Academic Publishers GmbH Printed in the United States
Autoimmunity. 1992. Vol. 14, pp. 155-158 Reprints available directly from the publisher Photocopying permitted by license only
HLA-DRIDQ GENE VARIATION IN NONGOITROUS AUTOIMMUNE THYROIDITIS AT THE SEROLOGICAL AND MOLECULAR LEVEL U. BOGNER, K. BADENHOOP*, H. PETERS, D. SCHMIEG, W.R. MAYR**, K.H.USADEL* and H. SCHLEUSENER Endocrine Department of the Medical Clinic, Klinikum Steglitz, Berlin, Germany, *Department of Endocrinology, Center of Internal Medicine, University Hospital, Frankfurt. Germany and **Institute of Transfusion Medicine, Technical University, Aachen, Germany
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(Received July 22,1992; infinal form October 9 , 1 9 9 2 ) The etiology of autoimmune diseases is multifactorial with genetic factors being an important prerequisite. There are two clinical manifestations of autoimmune thyroiditis: the goitrous form (Hashimoto:s thyroiditis) and the atrophic variant. which is characterized by hypothyroidism (primary myxoedema). Different genetic markers were assumed to be predisposing factors for the distinct clinical presentation. In the present study. we determined HLA A,B,C.DR,DQ alloantigens serologically and HLA-DQ by gene analysis in patients with nongoitrous autoimmune thyroiditis and randomly chosen controls. To verify the exact classifications, thyroid volume (median 5.85 ml) was measured by ultrasonography. HLA-DR5 was found in 16 of 36 (44%) patients with nongoitrous autoimmune thyroiditis and in only 26 of 175 controls (15%) (Pc=O.O018). There was a tendency towards a lower frequency of HLA-DR7 with 6% positivity in patients vs. 29% in controls (Pc=0.052). Regarding HLA-DQ, DQ7 was found in 17 of 35 patients (48%) vs. 21 of 98 controls (21%) (Pc=0.028) (relative risk 3.5). No other association was found with HLA-A.B.C and HLA-DR and -DQ. Our data indicate that the genetic susceptibility to autoimmune nongoitrous thyroiditis is closely associated to HLA-DRS and DQ7 and not distinct from goitrous disease. We conclude that factors other than genetic ones explain the different immunological and clinical manifestation of chronic lymphocytic thyroiditis. KEY WORDS: HLA-DR/DQ, nongoitrous autoimmune thyroiditis, primary myxwdema.
INTRODUCTION
assigned according to the 10th International Histocompatibility Workshop'.
There is now considerable evidence that chronic lymphocytic thyroiditis is an autoimmune disease. The etiology of such diseases is assumed to be multifactorial with predisposing genetic factors. In autoimmune thyroiditis, it has not yet been clarified why some patients develop a goiter with histologically intense lymphocytic infiltration (Hashimoto's thyroiditis) and others an atrophic gland (primary myxoedema) with minimal lymphocytic infiltration but extensive fibrosis. Different genetic factors have thus been assumed. Goitrous thyroiditis has been related to HLA-DR3, DR4 and DR5 and the atrophic variant to HLA-B8 and DR3"+. In the present study, we investigated autoimmune thyroiditis patients, in whom thyroid atrophy was demonstrated by ultrasonographic volumetry; HLA serology (A,B,C,DR,DQ) was applied for their HLAD specificities and restriction fragment length polymorphism (RFLP) analysis for DQ specificities, a probe being used for the DQBl gene and DQ being Correspondence to: Dr Ulrich Bogner, Endokrinologische Abteilung. Klinikum Steglitz, Hindenburgdamm 30, loo0 Berlin. Berlin 45. Germany.
I55
SUBJECTS AND METHODS Patients
The study included thirty-six patients (34 women, 2 men) from Berlin (Germany) with autoimmune thyroiditis. The diagnosis was based on elevated TSH values (TSH-IRMA>4 mU/l), and/or positive titers of microsomal ( > I : 100) and thyroglobulin (>1:40) antibodies and a typical hyporechoic thyroid ultrasonography. At the time of investigation thirty-three, previously hypothyroid patients, were euthyroid on longstanding I-thyroxine treatment (mean 113.6 pgld), two patients were hypothyroid without treatment and one patient euthyroid with significantly elevated autoantibody titers. The median titers of microsomal antibodies were 1:1600 (range 0-1:25600) and for thyroglobulin antibodies 1:80 (range 0-15120). Microsomal and thyroglobulin antibodies were determined by passive hemagglutination technique (Thymune M and Thymune T, Wellcome Diagnostics, Dartford, UK). Thyroid volume was calculated as described else-
I56
U. BOGNER ET AL.
thermore DNA amplified for the second exon of DQB 1 and hybridized with allele specific oligonucleotides confirmed those assignments (data not shown).
where*. Normal thyroid volume is 10-20 ml in women and 10-25 ml in men. Ultrasound volumetry of the thyroid showed a normal or atrophic gland (median 5.85 ml, range 1.7-13 ml) in all patients and a typical hypoechoic echopattern.
Statistical methods
Serological HLA and RFLP data were analysed using the chi-square test. Where indicated p-values (p,,,,) were corrected for the numbers of comparisons made. Relative risk (RR) were estimated as described by Woolf”. RFLP data for DQBl were correlated with serological data.
Autoimmunity Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
HLA serology HLA antigens of the A, B, and C loci were determined by the standard NIH microlymphocytotoxicity test, and HLA-DR and DQ 1-3 antigens by the double-color fluorescence technique using sera of the 8th and 9th International Histocompatibility Workshop. For HLAtyping clinically healthy Caucasian subjects served as controls (HLA A,B,C: n=840; HLA-DR: n=175; HLA-DQ: n=98).
RLFP studies Restriction fragment length polymorphism (RFLP) analysis was performed according to standard procedures recommended by the 10th International Histocompatibility Workshop Southern blotting protocol with some modifications’. In brief, DNA was prepared from peripheral blood, and 1Opg was digested with 20 units of restriction endonuclease TaqI and BamHI at the temperatures and buffer conditions specified by the manufacturers (Brthesda Research Laboratories, Amersham, USA). Digests were electrophoresed on a 0.8% agarose gel in TAE-buffer (40 mmol/l trisacetate, 1 mmol/l EDTA, pH 8), for 24 hr at 45 mA. Molecular weight markers-either HindIII-digested lambda DNA with HaeIII-digested phiX or 10th HLA Workshop Southern blot reference markers were run on each gel. The gels were soaked in 0.15 M HCL 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 hr with a radiolabeled cDNA probe for the DQBl gene in the following mixture: 50% deionised formamide, 0.1% Denhardt’s solution, SxSSPE, 1% SDS, 5% dextran sulphate and 200pglml salmon sperm DNA. The filters were washed twice in 2x SSPE at room temperature for 5 min. then in 2xSSPE, 0.5% SDS at 65°C for 15 min and finally in OSxSSPE at 65°C for 15 min. Using intensifying screens, they were then exposed for 3-10 days on Kodak-XAR-5 film at -80°C. Assignments of DQ-types by RFLP was done without knowledge of the D R D Q serotype. DQ specificities were assigned by analysing autoradiographs for specific DNA fragments (RFLP) known to characterise DNA sequence variation of those DQ specifi~ities~~l”. In order to distinguish HLA-DQ8 from DQ9 additional restriction enzyme digestion with MSP I was performed and southern blots were hybridized with the DRBl probe’.’’. Fur-
RESULTS The results of HLA A, B, and C typing did not significantly differ between patients with nongoitrous autoimmune thyroiditis and controls. The HLA-DR specificity DR5 was more frequent in patients than in controls: DR5 was found in 16 of 36 (44%) patients vs. 15% of normal subjects (x2=14.6; pc,,=0.0018) (Table 1). On the other hand, the HLA-DR7 antigen was less frequent, findings being positive in 2 of 36 patients (6%) with nongoitrous autoimmune thyroiditis vs. 29% in controls (x2=7.6; pc0,=O.052). Regarding the HLA DQ locus, patients showed a significantly higher frequency of DQ7 than controls: this marker was found in 17 of 35 patients (48%) but in only 21 of 98 controls (x’=8.03, pc,,=0.03) (Table 2). The other HLA DQ specificities did not differ between controls and patients. Table 1 Serologically defined HLA-DR markers in patients with nongoitrous autoimmune thyroiditis and controls ~
~~
Controls
HLA-DR Nongoitrous airtoimmune thyroiditis (n=36J
(n475J n (81
n fWJ 1
2 3 4 5 6 7
3 (8) 1 I(31)
10 (28) 8 (22) 16 (44) 12 (33) 2 (6)
46 (26) 65 (37) 41 (23) 43 (25) x’=14.6; Pc=0.0018 26(15) 30 (17) x2=7.6 Pc=O.O52 51 (29)
Table 2 DQ specificities defined by DQBl gene polymorphism in patients with nongoitrous autoimmune thyroiditis and controls DQ
Nongoirrous autoimmune thyroidiris (n=35) n f%J
Conrrols (n=98) n f%J
2 5 6
I2 (34) I I(31) I4 (40)
7 8
17 (49) 5 (14) I(3)
49 (50) 31 (32) 35 (36) 2 I (2 I ) I9 (19) 7 (7)
~
9
x’=8.03; Pc=O.O28
HLA MARKERS IN NONGOITROUS AUTOIMMUNE THYROIDITIS
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DISCUSSION
157
by Northern blot analysis2’. However, a direct antigen-presenting function distinct from DR has yet to be shown. Since nearly half of the patients have neither the DQ7 nor DR5 high-risk marker, putative disease-susceptibility gene(s) might be located close to the DNA region analyzed so far. DR5 and DQ7 might therefore represent immunogenetic markers on a susceptible HLA haplotype that is closely linked to another gene locus. Recently, Badenhoop et al. described a strong association of HLA DQ7 with goitrous thyroiditis (relative risk 4.7)4. Our findings of the same genetic association in atrophic thyroiditis provide evidence that the manifestation of either the atrophic or the goitrous type of autoimmune thyroiditis is not linked to different genetic factors. After the onset of the autoimmune process against the thyroid in affected patients, factors other than genetic ones will determine the pathogenetic course of the disease. TSHreceptor-binding and -blocking antibodies are often more closely associated with atrophic disease, although their incidence is too low to explain conclusively the different clinical presentati~n”-’~.There is evidence that the occurrence of thyroid-specific cytotoxic antibodies might be the decisive step for induction of gland atrophy, since they are significantly more often associated with atrophic than with goitrous autoimmune thyroiditis (own observation, submitted for publication). In summary, in our study populations genetic markers of the HLA-DR/DQ region in autoimmune thyroiditis are similarly distributed in atrophic and hypertrophic disease4. It needs to be shown that this is applicable for patient populations from other areas5s6. From our studies we have evidence that HLA molecules or related products might confer susceptibility to thyroid autoimmunity in general whereas other factors determine the various forms of pathophysiology and clinical manifestation.
It has not yet been clarified whether goitrous and atrophic autoimmune thyroiditis are variants of the same disease with primary myxoedema as the end stage of goitrous chronic lymphocytic thyroiditis. Due to the specific association with genetic markers and the different prevalence of autoantibodies in these diseases, Doniach considered Hashimoto’s thyroiditis and primary myxoedema to be separate entities of autoimmune thyroiditis”. Conflicting evidence exists as to the HLA associations in Hashimoto’s thyroiditis and primary myxoedema with some studies supporting a predominance of HLA-DR4 and DR5 in the former and HLA-DR3 in the latter’“. Our data on these genetic markers demonstrate a significantly higher association with the HLA-DRS locus in atrophic autoimmune thyroiditis, which is identical to data reported in goitrous Hashimoto’s The fact that these findings are at variance with earlier data could be partly due to 1) newly described HLA-specificities and methods of HLA-assignment and 2) the introduction of ultrasonography, which allows exact thyroid volumetry and obviates misclassification due to inaccurate estimation by inspection and palpation. Furthermore, we present here for the first time a full description of RFLP-defined DQ-types in primary myxoedema according to the new nomenclature established by the 10th International Histocompatibility Workshop’. RFLP analysis reveals the DQ7 specificity as another DR5-linked marker with a strong association. It is not known whether the main association in primary myxoedema is due to DR5 or DQ7 or both. In contrast to Hashimoto’s thyroiditis where DR4 and DR5 associations are explained by DQ7, in primary myxoedema no other DR specificity showed an association4. DQ specificities are encoded by the DQAl and DQBl genes that harbor the variable regions for the DQ alleles, with hypervariable regions coding for the first Particular nucleic-acid codons differ in DQ7 from those in other DQ types”. At codon 45 a References glutamic acid is unique to DQ7, determines the recogI . Farid NR, Thompson C. HLA and autoimmune endocrine disnition of DQ7 by the monoclonal antibody TAlO and ease 1985; M o l Biol Med 1986; 3: 85-97 contributes to the steric structure recognized by serol2. Weissel M, Hofer R, Zasmeta H, Mayr WR. HLA-DR and ogY’6,17In analogy to the crystallized HLA-A2 molHashimoto’s thyroiditis. Tissue Antigens 1980; 16: 256-257 ecule, HLA class I1 molecules with their three-dimen3. Farid NR, Balacs C. Immunogenetics of thyroiditis and thyroid sional structure can be used for peptide binding carcinoma. In: Farid NR, ed. Immunogenetics of endocrine disorders. New York: Alan R. Liss Inc, 1988; 267-307 model^'^^'^. According to this model, the unique glu4. Badenhoop K, Schwarz G, Walfish PG. Drummond V. Usadel tamic acid of DQ7 would be situated at the bottom of KH. Bottazzo GF. Susceptibility to thyroid autoimmune disthe P-pleated sheet of the DQ molecule, thus facing ease: molecular analysis of HLA-D region genes identifies new the peptide binding site““’. The DQ7 molecule could markers for goitrous Hashimoto’s thyroiditis. J Clin EndocrinolMrruh 1990: 71: 1131-1137 preferentially present antigenic peptides to the T-cell 5 . Tandon N. Zhang L. Weetman AP. HLA associations with and/or facilitate a breakdown of tolerance towards the Hashirnoto’s thyroiditis. Clin Endacrinol 199 1; 34: 383-396 thyroid autoantigen. 6. Jenkins D, Penny MA, Fletcher JA, er ul. HLA class I1 gene DQ molecules can also be expressed in thyroid cells polymorphism contributes little to Hashimoto’s thyroiditis. Clin Endocrinol 1992: 37: 141-145 after stimulation with gamma-interferon, as detected
.
Autoimmunity Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
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U. BOGNER ET AL.
7. Dupont B. Nomenclature for factors of the HLA system, 1987. Hum Immunol 1989; 26: 3-14 8. Gutekunst R. Smolarek H, Hasenpusch U, er a/. Goitre epidemiology: thyroid volume, iodine excretion, thyroglobulin and thyrotropin in Germany and Sweden. Acra Endocrinol (Copenh) 1986; 112: 494-501 9. Martell M, Marcadet A, Cohen D. HLA Class I1 typing by the workshop defined RFLP specificities in unrelated individuals. In: Dupont 9, ed. Immunobiology of HLA. Volume I : Histocompatibiliry Tesring 1987. New York: Springer-Verlag. 1989; 943-952 10. Marcadet A, O'Connell P. Cohen D. Standardized Southern blot workshop technique. In: Dupont B. ed. Immunobiology of HLA. Volume I : Hisrocompatibiliry Testing 1987. New York: Springer-Verlag. 1989; 553-560 1 I . Woolf B. On estimating the relation between blood group and disease. Ann Hum Genet 1955; 19: 25 1-255 12. Doniach D. Hashimoto's thyroiditis and primary myxoedema viewed as separate entities. Europ J Clin Invesr 1981; 11: 245-247 13. Todd JA, Bell JI. McDevitt HO. HLA-DQPgene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Narure 1987; 329: 599-604 14. Horn GT, Bugawan TL, Long CM, Erlich HA. Allelic sequence variation of the HLA-DQ loci: Relationship to serology and to insulin-dependent diabetes susceptibility. Proc Nail Acad Sci USA 1988; 85: 60 12-60 16 15. Todd JA. Bell JL, McDevitt HO. A molecular basis for genetic susceptibility to insulin-dependent diabetes mellitus. Trends Genet 1988; 4: 129-134 16. Kwok WW, Lotshaw C. Milner ECB, Knitter-Jack N, Nepom
17. 18. 19.
20.
2 1.
22.
23.
GT. Mutational analysis of the HLA-DQ3.2 insulin-dependent diabetes mellitus susceptibility gene. Proc Nut1 Acad Sci USA 1989; 86: 1027-1030 Radka SF, Nelson KA. Johnston JV. HLA-DQw3-related determinants: Analysis of subunit and spatial relationships. Hum Immunol 1989; 25: 225-236 Bjorkman PJ, Saper MA, Samraoui B, Bennett WS. Strominger JL. Wiley DC. Structure of the human class I histocompatibility antigen. HLA-A2. Narure 1987; 329: 50651 I Bjorkman PJ. Saper MA, Samraoui B, Bennett WS, Strominger JL. Wiley DC. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Narure 1987; 329: 512-516 Badenhoop KWJ. Buscema M, Belfiore A. Pujol-Borrell R, Trowsdale J, Bottazzo GF. Expression of HLA-D subregion genes in thyroid follicular cells transfected with SV-40. In: Dupont B, ed. Immunobiology of HLA. Volume II: Immunogenetics and Hisrocompatibiliry. New York: Springer-Verlag. 1989: 358-360 Tamaki H. Amino N. Kimura M, Hidaka Y, Takeoda K. Miyai K. Low prevalence of thyrotropin receptor antibody in primary hypothyroidism in Japan. J Clin Endocrinol Merab 1990 71: 1382-1386 Steel NR, Weightman DR, Taylor JJ, Kendall-Taylor P. Antibodies that block stimulation by TSH or TSAb of thyroid hormone secretion in primary hypothyroidism. Auroimmunity 1988; 1: 81-90 Arikawa K. Ichikawa Y. Yoshida T. et al. Blocking type antithyrotropin receptor antibody in patients with nongoitrous hypothyroidism: its incidence and characteristics of action. J Clin Endocrinol Metah 1985; 60: 953-959
Autoimmunity. 1992. Vol. 14, pp. 155-158 Reprints available directly from the publisher Photocopying permitted by license only
HLA-DRIDQ GENE VARIATION IN NONGOITROUS AUTOIMMUNE THYROIDITIS AT THE SEROLOGICAL AND MOLECULAR LEVEL U. BOGNER, K. BADENHOOP*, H. PETERS, D. SCHMIEG, W.R. MAYR**, K.H.USADEL* and H. SCHLEUSENER Endocrine Department of the Medical Clinic, Klinikum Steglitz, Berlin, Germany, *Department of Endocrinology, Center of Internal Medicine, University Hospital, Frankfurt. Germany and **Institute of Transfusion Medicine, Technical University, Aachen, Germany
Autoimmunity Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
(Received July 22,1992; infinal form October 9 , 1 9 9 2 ) The etiology of autoimmune diseases is multifactorial with genetic factors being an important prerequisite. There are two clinical manifestations of autoimmune thyroiditis: the goitrous form (Hashimoto:s thyroiditis) and the atrophic variant. which is characterized by hypothyroidism (primary myxoedema). Different genetic markers were assumed to be predisposing factors for the distinct clinical presentation. In the present study. we determined HLA A,B,C.DR,DQ alloantigens serologically and HLA-DQ by gene analysis in patients with nongoitrous autoimmune thyroiditis and randomly chosen controls. To verify the exact classifications, thyroid volume (median 5.85 ml) was measured by ultrasonography. HLA-DR5 was found in 16 of 36 (44%) patients with nongoitrous autoimmune thyroiditis and in only 26 of 175 controls (15%) (Pc=O.O018). There was a tendency towards a lower frequency of HLA-DR7 with 6% positivity in patients vs. 29% in controls (Pc=0.052). Regarding HLA-DQ, DQ7 was found in 17 of 35 patients (48%) vs. 21 of 98 controls (21%) (Pc=0.028) (relative risk 3.5). No other association was found with HLA-A.B.C and HLA-DR and -DQ. Our data indicate that the genetic susceptibility to autoimmune nongoitrous thyroiditis is closely associated to HLA-DRS and DQ7 and not distinct from goitrous disease. We conclude that factors other than genetic ones explain the different immunological and clinical manifestation of chronic lymphocytic thyroiditis. KEY WORDS: HLA-DR/DQ, nongoitrous autoimmune thyroiditis, primary myxwdema.
INTRODUCTION
assigned according to the 10th International Histocompatibility Workshop'.
There is now considerable evidence that chronic lymphocytic thyroiditis is an autoimmune disease. The etiology of such diseases is assumed to be multifactorial with predisposing genetic factors. In autoimmune thyroiditis, it has not yet been clarified why some patients develop a goiter with histologically intense lymphocytic infiltration (Hashimoto's thyroiditis) and others an atrophic gland (primary myxoedema) with minimal lymphocytic infiltration but extensive fibrosis. Different genetic factors have thus been assumed. Goitrous thyroiditis has been related to HLA-DR3, DR4 and DR5 and the atrophic variant to HLA-B8 and DR3"+. In the present study, we investigated autoimmune thyroiditis patients, in whom thyroid atrophy was demonstrated by ultrasonographic volumetry; HLA serology (A,B,C,DR,DQ) was applied for their HLAD specificities and restriction fragment length polymorphism (RFLP) analysis for DQ specificities, a probe being used for the DQBl gene and DQ being Correspondence to: Dr Ulrich Bogner, Endokrinologische Abteilung. Klinikum Steglitz, Hindenburgdamm 30, loo0 Berlin. Berlin 45. Germany.
I55
SUBJECTS AND METHODS Patients
The study included thirty-six patients (34 women, 2 men) from Berlin (Germany) with autoimmune thyroiditis. The diagnosis was based on elevated TSH values (TSH-IRMA>4 mU/l), and/or positive titers of microsomal ( > I : 100) and thyroglobulin (>1:40) antibodies and a typical hyporechoic thyroid ultrasonography. At the time of investigation thirty-three, previously hypothyroid patients, were euthyroid on longstanding I-thyroxine treatment (mean 113.6 pgld), two patients were hypothyroid without treatment and one patient euthyroid with significantly elevated autoantibody titers. The median titers of microsomal antibodies were 1:1600 (range 0-1:25600) and for thyroglobulin antibodies 1:80 (range 0-15120). Microsomal and thyroglobulin antibodies were determined by passive hemagglutination technique (Thymune M and Thymune T, Wellcome Diagnostics, Dartford, UK). Thyroid volume was calculated as described else-
I56
U. BOGNER ET AL.
thermore DNA amplified for the second exon of DQB 1 and hybridized with allele specific oligonucleotides confirmed those assignments (data not shown).
where*. Normal thyroid volume is 10-20 ml in women and 10-25 ml in men. Ultrasound volumetry of the thyroid showed a normal or atrophic gland (median 5.85 ml, range 1.7-13 ml) in all patients and a typical hypoechoic echopattern.
Statistical methods
Serological HLA and RFLP data were analysed using the chi-square test. Where indicated p-values (p,,,,) were corrected for the numbers of comparisons made. Relative risk (RR) were estimated as described by Woolf”. RFLP data for DQBl were correlated with serological data.
Autoimmunity Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
HLA serology HLA antigens of the A, B, and C loci were determined by the standard NIH microlymphocytotoxicity test, and HLA-DR and DQ 1-3 antigens by the double-color fluorescence technique using sera of the 8th and 9th International Histocompatibility Workshop. For HLAtyping clinically healthy Caucasian subjects served as controls (HLA A,B,C: n=840; HLA-DR: n=175; HLA-DQ: n=98).
RLFP studies Restriction fragment length polymorphism (RFLP) analysis was performed according to standard procedures recommended by the 10th International Histocompatibility Workshop Southern blotting protocol with some modifications’. In brief, DNA was prepared from peripheral blood, and 1Opg was digested with 20 units of restriction endonuclease TaqI and BamHI at the temperatures and buffer conditions specified by the manufacturers (Brthesda Research Laboratories, Amersham, USA). Digests were electrophoresed on a 0.8% agarose gel in TAE-buffer (40 mmol/l trisacetate, 1 mmol/l EDTA, pH 8), for 24 hr at 45 mA. Molecular weight markers-either HindIII-digested lambda DNA with HaeIII-digested phiX or 10th HLA Workshop Southern blot reference markers were run on each gel. The gels were soaked in 0.15 M HCL 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 hr with a radiolabeled cDNA probe for the DQBl gene in the following mixture: 50% deionised formamide, 0.1% Denhardt’s solution, SxSSPE, 1% SDS, 5% dextran sulphate and 200pglml salmon sperm DNA. The filters were washed twice in 2x SSPE at room temperature for 5 min. then in 2xSSPE, 0.5% SDS at 65°C for 15 min and finally in OSxSSPE at 65°C for 15 min. Using intensifying screens, they were then exposed for 3-10 days on Kodak-XAR-5 film at -80°C. Assignments of DQ-types by RFLP was done without knowledge of the D R D Q serotype. DQ specificities were assigned by analysing autoradiographs for specific DNA fragments (RFLP) known to characterise DNA sequence variation of those DQ specifi~ities~~l”. In order to distinguish HLA-DQ8 from DQ9 additional restriction enzyme digestion with MSP I was performed and southern blots were hybridized with the DRBl probe’.’’. Fur-
RESULTS The results of HLA A, B, and C typing did not significantly differ between patients with nongoitrous autoimmune thyroiditis and controls. The HLA-DR specificity DR5 was more frequent in patients than in controls: DR5 was found in 16 of 36 (44%) patients vs. 15% of normal subjects (x2=14.6; pc,,=0.0018) (Table 1). On the other hand, the HLA-DR7 antigen was less frequent, findings being positive in 2 of 36 patients (6%) with nongoitrous autoimmune thyroiditis vs. 29% in controls (x2=7.6; pc0,=O.052). Regarding the HLA DQ locus, patients showed a significantly higher frequency of DQ7 than controls: this marker was found in 17 of 35 patients (48%) but in only 21 of 98 controls (x’=8.03, pc,,=0.03) (Table 2). The other HLA DQ specificities did not differ between controls and patients. Table 1 Serologically defined HLA-DR markers in patients with nongoitrous autoimmune thyroiditis and controls ~
~~
Controls
HLA-DR Nongoitrous airtoimmune thyroiditis (n=36J
(n475J n (81
n fWJ 1
2 3 4 5 6 7
3 (8) 1 I(31)
10 (28) 8 (22) 16 (44) 12 (33) 2 (6)
46 (26) 65 (37) 41 (23) 43 (25) x’=14.6; Pc=0.0018 26(15) 30 (17) x2=7.6 Pc=O.O52 51 (29)
Table 2 DQ specificities defined by DQBl gene polymorphism in patients with nongoitrous autoimmune thyroiditis and controls DQ
Nongoirrous autoimmune thyroidiris (n=35) n f%J
Conrrols (n=98) n f%J
2 5 6
I2 (34) I I(31) I4 (40)
7 8
17 (49) 5 (14) I(3)
49 (50) 31 (32) 35 (36) 2 I (2 I ) I9 (19) 7 (7)
~
9
x’=8.03; Pc=O.O28
HLA MARKERS IN NONGOITROUS AUTOIMMUNE THYROIDITIS
Autoimmunity Downloaded from informahealthcare.com by Nyu Medical Center on 02/02/15 For personal use only.
DISCUSSION
157
by Northern blot analysis2’. However, a direct antigen-presenting function distinct from DR has yet to be shown. Since nearly half of the patients have neither the DQ7 nor DR5 high-risk marker, putative disease-susceptibility gene(s) might be located close to the DNA region analyzed so far. DR5 and DQ7 might therefore represent immunogenetic markers on a susceptible HLA haplotype that is closely linked to another gene locus. Recently, Badenhoop et al. described a strong association of HLA DQ7 with goitrous thyroiditis (relative risk 4.7)4. Our findings of the same genetic association in atrophic thyroiditis provide evidence that the manifestation of either the atrophic or the goitrous type of autoimmune thyroiditis is not linked to different genetic factors. After the onset of the autoimmune process against the thyroid in affected patients, factors other than genetic ones will determine the pathogenetic course of the disease. TSHreceptor-binding and -blocking antibodies are often more closely associated with atrophic disease, although their incidence is too low to explain conclusively the different clinical presentati~n”-’~.There is evidence that the occurrence of thyroid-specific cytotoxic antibodies might be the decisive step for induction of gland atrophy, since they are significantly more often associated with atrophic than with goitrous autoimmune thyroiditis (own observation, submitted for publication). In summary, in our study populations genetic markers of the HLA-DR/DQ region in autoimmune thyroiditis are similarly distributed in atrophic and hypertrophic disease4. It needs to be shown that this is applicable for patient populations from other areas5s6. From our studies we have evidence that HLA molecules or related products might confer susceptibility to thyroid autoimmunity in general whereas other factors determine the various forms of pathophysiology and clinical manifestation.
It has not yet been clarified whether goitrous and atrophic autoimmune thyroiditis are variants of the same disease with primary myxoedema as the end stage of goitrous chronic lymphocytic thyroiditis. Due to the specific association with genetic markers and the different prevalence of autoantibodies in these diseases, Doniach considered Hashimoto’s thyroiditis and primary myxoedema to be separate entities of autoimmune thyroiditis”. Conflicting evidence exists as to the HLA associations in Hashimoto’s thyroiditis and primary myxoedema with some studies supporting a predominance of HLA-DR4 and DR5 in the former and HLA-DR3 in the latter’“. Our data on these genetic markers demonstrate a significantly higher association with the HLA-DRS locus in atrophic autoimmune thyroiditis, which is identical to data reported in goitrous Hashimoto’s The fact that these findings are at variance with earlier data could be partly due to 1) newly described HLA-specificities and methods of HLA-assignment and 2) the introduction of ultrasonography, which allows exact thyroid volumetry and obviates misclassification due to inaccurate estimation by inspection and palpation. Furthermore, we present here for the first time a full description of RFLP-defined DQ-types in primary myxoedema according to the new nomenclature established by the 10th International Histocompatibility Workshop’. RFLP analysis reveals the DQ7 specificity as another DR5-linked marker with a strong association. It is not known whether the main association in primary myxoedema is due to DR5 or DQ7 or both. In contrast to Hashimoto’s thyroiditis where DR4 and DR5 associations are explained by DQ7, in primary myxoedema no other DR specificity showed an association4. DQ specificities are encoded by the DQAl and DQBl genes that harbor the variable regions for the DQ alleles, with hypervariable regions coding for the first Particular nucleic-acid codons differ in DQ7 from those in other DQ types”. At codon 45 a References glutamic acid is unique to DQ7, determines the recogI . Farid NR, Thompson C. HLA and autoimmune endocrine disnition of DQ7 by the monoclonal antibody TAlO and ease 1985; M o l Biol Med 1986; 3: 85-97 contributes to the steric structure recognized by serol2. Weissel M, Hofer R, Zasmeta H, Mayr WR. HLA-DR and ogY’6,17In analogy to the crystallized HLA-A2 molHashimoto’s thyroiditis. Tissue Antigens 1980; 16: 256-257 ecule, HLA class I1 molecules with their three-dimen3. Farid NR, Balacs C. Immunogenetics of thyroiditis and thyroid sional structure can be used for peptide binding carcinoma. In: Farid NR, ed. Immunogenetics of endocrine disorders. New York: Alan R. Liss Inc, 1988; 267-307 model^'^^'^. According to this model, the unique glu4. Badenhoop K, Schwarz G, Walfish PG. Drummond V. Usadel tamic acid of DQ7 would be situated at the bottom of KH. Bottazzo GF. Susceptibility to thyroid autoimmune disthe P-pleated sheet of the DQ molecule, thus facing ease: molecular analysis of HLA-D region genes identifies new the peptide binding site““’. The DQ7 molecule could markers for goitrous Hashimoto’s thyroiditis. J Clin EndocrinolMrruh 1990: 71: 1131-1137 preferentially present antigenic peptides to the T-cell 5 . Tandon N. Zhang L. Weetman AP. HLA associations with and/or facilitate a breakdown of tolerance towards the Hashirnoto’s thyroiditis. Clin Endacrinol 199 1; 34: 383-396 thyroid autoantigen. 6. Jenkins D, Penny MA, Fletcher JA, er ul. HLA class I1 gene DQ molecules can also be expressed in thyroid cells polymorphism contributes little to Hashimoto’s thyroiditis. Clin Endocrinol 1992: 37: 141-145 after stimulation with gamma-interferon, as detected
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7. Dupont B. Nomenclature for factors of the HLA system, 1987. Hum Immunol 1989; 26: 3-14 8. Gutekunst R. Smolarek H, Hasenpusch U, er a/. Goitre epidemiology: thyroid volume, iodine excretion, thyroglobulin and thyrotropin in Germany and Sweden. Acra Endocrinol (Copenh) 1986; 112: 494-501 9. Martell M, Marcadet A, Cohen D. HLA Class I1 typing by the workshop defined RFLP specificities in unrelated individuals. In: Dupont 9, ed. Immunobiology of HLA. Volume I : Histocompatibiliry Tesring 1987. New York: Springer-Verlag. 1989; 943-952 10. Marcadet A, O'Connell P. Cohen D. Standardized Southern blot workshop technique. In: Dupont B. ed. Immunobiology of HLA. Volume I : Hisrocompatibiliry Testing 1987. New York: Springer-Verlag. 1989; 553-560 1 I . Woolf B. On estimating the relation between blood group and disease. Ann Hum Genet 1955; 19: 25 1-255 12. Doniach D. Hashimoto's thyroiditis and primary myxoedema viewed as separate entities. Europ J Clin Invesr 1981; 11: 245-247 13. Todd JA, Bell JI. McDevitt HO. HLA-DQPgene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Narure 1987; 329: 599-604 14. Horn GT, Bugawan TL, Long CM, Erlich HA. Allelic sequence variation of the HLA-DQ loci: Relationship to serology and to insulin-dependent diabetes susceptibility. Proc Nail Acad Sci USA 1988; 85: 60 12-60 16 15. Todd JA. Bell JL, McDevitt HO. A molecular basis for genetic susceptibility to insulin-dependent diabetes mellitus. Trends Genet 1988; 4: 129-134 16. Kwok WW, Lotshaw C. Milner ECB, Knitter-Jack N, Nepom
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GT. Mutational analysis of the HLA-DQ3.2 insulin-dependent diabetes mellitus susceptibility gene. Proc Nut1 Acad Sci USA 1989; 86: 1027-1030 Radka SF, Nelson KA. Johnston JV. HLA-DQw3-related determinants: Analysis of subunit and spatial relationships. Hum Immunol 1989; 25: 225-236 Bjorkman PJ, Saper MA, Samraoui B, Bennett WS. Strominger JL. Wiley DC. Structure of the human class I histocompatibility antigen. HLA-A2. Narure 1987; 329: 50651 I Bjorkman PJ. Saper MA, Samraoui B, Bennett WS, Strominger JL. Wiley DC. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Narure 1987; 329: 512-516 Badenhoop KWJ. Buscema M, Belfiore A. Pujol-Borrell R, Trowsdale J, Bottazzo GF. Expression of HLA-D subregion genes in thyroid follicular cells transfected with SV-40. In: Dupont B, ed. Immunobiology of HLA. Volume II: Immunogenetics and Hisrocompatibiliry. New York: Springer-Verlag. 1989: 358-360 Tamaki H. Amino N. Kimura M, Hidaka Y, Takeoda K. Miyai K. Low prevalence of thyrotropin receptor antibody in primary hypothyroidism in Japan. J Clin Endocrinol Merab 1990 71: 1382-1386 Steel NR, Weightman DR, Taylor JJ, Kendall-Taylor P. Antibodies that block stimulation by TSH or TSAb of thyroid hormone secretion in primary hypothyroidism. Auroimmunity 1988; 1: 81-90 Arikawa K. Ichikawa Y. Yoshida T. et al. Blocking type antithyrotropin receptor antibody in patients with nongoitrous hypothyroidism: its incidence and characteristics of action. J Clin Endocrinol Metah 1985; 60: 953-959
