mhc-extended haplotypes in families of patients with grave's disease

MHC-Extended Haplotypes in Families of Patients with Graves' Disease

Suvina Ratanachaiyavong, Linda Lloyd, Christopher Darke, and Alan M. McGregor

ABSTRACT: MHC-extended haplotypes were investi- gated in multiplex families of patients with hyperthyroid GD. Using a combination of both phenotypic (serology and protein electrophoresis) and genotypic (DNA-RFLP) markers, 159 MHC-extended haplotypes extending from HLA-A across the MHC class III (C2, Bf, C4A, and C4B) toward the HLA-DR/DQ complex were deduced from 217 (51 and 166 affected and unaffected) members of 21 families of patients with GD. Thyroid autoantibodies were measured and found positive in 27.1% of 166 clinically euthyroid unaffected members. Extended haplotypes were classified into four categories--affected (n = 40), Aff/Ab+ve (shared haplotype between affected and Ab + ve members, n -- 31), Ab + ve (n = 29), and Ab - ve (n = 59)--based on the presence and absence of these haplotypes in 51 affected members with GD and 45 and 121 unaffected members who were respectively positive and negative for thyroid autoantibodies. Five recombina-

tions were detected: three were found between HLA-A and B and two between HLA-B and the MHC class III. No recombination was found between or within the MHC class III and class II complex. Though the HLA-DR17 (DRy171 and DRy172) allele was found to be significantly increased in both the affected and the Aff/Ab + ve when compared with the A b - v e haplotypes (p < 0.042 and p < 0.018), the frequency of the HLA-B8, 2.7-kb SstI- 4.5-kb TaqI/C2 Bf*S, 6.4-kb TaqI/C4A*QOC4B*I, HLA-DR/~171/DQ~2-DQ~2a extended haplotype was found to be significantly increased only in the affected haplotype (p < 0.05). These results suggest that while HLA-DR17 is a susceptibility allele shared between GD and individuals with positive thyroid autoantibodies, the HLA-B8, 2.7-kb SstI-4.5-kb TaqI/5'-3'C2 Bf*S, 6.4-kb TaqI/C4A*QOB*I, DR~171/DQ~2-DQ32a is a disease susceptibility-extended haplotype for Graves' disease. Human Immunology 36, 99-• 11 (1993)

ABBREVIATIONS A b - v e thyroid autoantibody negative Ab + ve thyroid autoantibody positive Aft affected Bf factor B bp base pair DNA-RFLP DNA-restriction fragment length

polymorphism GD Graves' disease

HLA human leukocyte associated (antigen)

IDDM insulin-dependent diabetes mellitus

LD linkage disequilibrium MHC major histocompatibility

complex

I N T R O D U C T I O N

Graves' disease (GD) is a common organ-specific autoimmune disease characterized immunologically by the presence of autoantibodies against the thyrotropin re-

From the Department of Medicine (S.R., L.L., A.M.M.), King's College School of Medicine, London," and the Tissue Typing Laboratory (C.D.), Blood Transfusion Centre, Cardiff Wales, UK.

Address reprint requests to Dr. S. Ratanachaiyavong, Department of Biomedical Sciences, Faculty of Medicine, Prince of Songkla University, Hat-Yai Songkla 90112, Thailand.

Received November 5, 1991; accepted October 30, 1992.

ceptor, thyroid peroxidase (thyroid microsomal antigen), thyroglobulin, and possibly several undefined thy- roidal antigens, in conjunction with intrathyroidal lymphocytic infiltration.

The association of the major histocompatibility complex (MHC) antigens, human leukocyte associated antigen (HLA)-B8 and DR3(17), with GD in Caucasoid populations has been well documented [ 1]. Though the association of HLA-DR3 with GD is not particularly strong, this association has been confirmed at the genotypic (DNA) level [2]. Unlike insulin-dependent diabe-

Human Immunology 36, 99-l 11 (1993) 99 © American Society for Histocompatibility and lmmunogenetics, 1993 0198-8859/93/$6.00

100 S. Ratanachaiyavong et al.

T A B L E 1 Distribution of number of affected and unaffected individuals with number of extended haplotypes deduced from each family of patients with Graves' disease

Number of individuals Number of extended haplotypes

Family Aft Ab + ve Ab - ve Total Aft Aff/Ab + Ab + ve Ab - ve Total

1) A 8 7 (33.3) 14 29 2 3 5 5 15 2) B 4 9 (37.5) 15 28 1 4 4 5 14 3 / C 4 9 (56.3) 7 20 0 6 4 4 14 4) D 3 2 (22.2) 7 12 1 3 1 3 8 5) E 3 1 (11.1) 8 12 4 1 1 4 10 6) F 3 1 (50.0) 1 5 3 1 1 0 5 7) G 3 l (16.7) 5 9 4 1 1 0 6 8) H 2 3 (26.4) 9 14 0 3 l 3 7 9/ 1 2 3 (37.5) 5 10 0 4 1 3 8

10) J 2 1 (20.0) 4 7 2 1 1 3 7 11 /K 2 1 (20.0) 4 7 3 0 2 1 6 12) L 2 0 - - 4 6 3 0 0 2 5 13) M 2 0 - 1 3 3 0 0 1 4 141 N 2 0 - 4 6 3 0 0 3 6 15) O 2 0 - 4 6 3 0 0 3 6 16) P 2 0 - - 3 5 2 0 0 4 6 17) Q 1 2 (66.7) 1 4 0 2 1 1 4 18) R 1 3 (30.0) 7 11 1 1 3 4 9 19) S 1" 1 (20.0) 4 6 2 0 2 2 6 20) T 1 1 (11.1) 8 10 1 1 1 4 7 21) U 1 0 - 6 7 2 0 0 4 6

Total 51 45 (27.1) 121 217 40 31 29 59 159

Percentage of positive thyroid autoantibody is shown in parentheses. *Blood sample from the second affected member was not available. Aft, affected haplotypes deduced from affected members excluding haplotypes shared with Ab + ve members; Aff/Ab +, haplotypes shared between affected and Ab + ve members regardless of sharing with A b - ve members; Ab + ve, haplotypes shared between Ab + ve members regardless of sharing with A b - ve members; and A b - ve, haplotypes shared between A b - ve members excluding any haplotypes shared with either affected or Ab + ve members.

tes mellitus (IDDM), which is likely to affect the younger age group (i.e., less than 25 years of age), GD can occur over a wide age range, with the mean age of diagnosis being 48 years. Females are more likely to be affected by the GD than are males, with an approximate ratio of 10 : 1 [3]. A family history of autoimmune thyroid disease is well recognized in approximately 20% of the patients with GD, suggesting a genetic contribution toward development of the disease in some but not all of the families. Are there any genetic differences between and within the families that have multiple members affected? Can any genetic marker be identified that could be used to predict the future development of GD in family members who have not yet been affected by the disease? We have sought to answer these questions.

The human MHC is highly polymorphic at both the genotypic and phenotypic levels. The MHC class I, II, and III genes encode transmembrane glycoproteins and several serum complement components that are primar- ily involved in cellular and humoral immune responses. The complexity of the MHC, its genomic organization, and physical distance map have recently been established

[4] and seem to be the best immunogenetic markers that have been explored so far. To address the above questions, we decided to use both phenotypic and genotypic approaches to investigate extensively families of patients having one or more members affected with GD.

M A T E R I A L S A N D M E T H O D S

Families. A total of 217 blood samples were collected from 2 -3 generations of both affected and unaffected members of 21 British Caucasoid families from England and Wales. There are eight, four, three, two, and one members in one, two, four, ten, and four families affected with GD, respectively (Table 1).

Peripheral blood mononuclear cells were freshly isolated from heparinized blood samples and stored in liq- uid nitrogen for the HLA-A and -B serologic typing. Fresh frozen plasma samples were stored at - 70°C for factor B (Bf) and C4 protein typing. Serum samples were stored at - 20°C for measurement of thyroid autoantibodies. D N A was extracted from EDTA blood samples and stored at - 20°C for further restriction digests.

MHC-Extended Haplotypes in Graves' Disease 101

HLA typing. HLA-A and HLA-B were determined se- rologically by the standard lymphocyte microcytotox- icity test [5] using a panel of well-characterized antisera used for tissue typing in the Tissue Typing Department, Blood Transfusion Centre, Rhydlafar, Cardiff.

Factor-B typing. Plasma samples were subjected to 0.8% agarose gel electrophoresis and visualized by Coomassie staining after immunofixation with goat-anti-human Bf antiserum (Atlantic Antibodies) as described [6].

C4 protein typing. Plasma samples were desialated with neuraminidase enzyme from Clostridium perfringens type VI (Sigma, UK) at a concentration of 5 mU/~l of plasma at room temperature overnight. C4 structural variants were detected by agarose gel electrophoresis (agarose type II; Sigma) and immunofixation with rabbit antiserum against human C4 (Behring Diagnostic, Hoechst UK) as described by Awdeh and Alper [7]. The designa- tion of C4 phenotypes is based on "Statement on the Nomenclature of Human C4 Allotypes" [8].

Thyroid autoantibodies measurement. The thyroid microsomal and thyroglobulin antibodies were detected in all serum samples by using an enzyme-linked immunosor- bent assay as previously described [9].

DNA extraction and hybridization. Genomic DNA was prepared from whole blood samples by using sucrose lysis buffer as previously described [ 10]. Approximately 3-5 tzg DNA was digested with restriction endonucle- ases SstI and TaqI (Gibco-BRL) under the conditions recommended by the manufacturer, size-fractionated by gel electrophoresis in 0.9% agarose-1 × TBE buffer for SstI and 0.7% agarose-1 × TAE buffer for TaqI, and transferred to nylon membranes [ 11]. Hybridization was carried out with appropriate 32P-labeled probes, at 65°C, overnight under conditions previously described [10].

DNAprobes. All DNA probes were prepared by plasmid miniprep using the alkali lysis method [12]:

1. The C2 probe is a 300-base-pair (bp) BamHI-KpnI fragment derived from the genomic clone pG850, which detects the SstI polymorphism at the 5' end of the C2 locus [13].

2. The Bf probe is a 660-bp ClaI-BamHI fragment derived from cDNA clone pFB3b, which detects the TaqI polymorphism at the 3' end of the C2 gene [14].

3. The C4 probe used in this study is a 5.5-kb full-length C4 cDNA probe (pAT-A) described by Belt and colleagues [ 15]. The HLA-DRB 1 probe is a locus-specific 517-bp PstI .

fragment (pRTV 1) originally derived from pDR-/3-2 probe [16].

5. The HLA-DQA1 probe is a full-length DQ~ (pII-~- 5) probe [17].

6. The HLA-DQB1 probe is a 627-bp AvaI fragment from pII-/3-1 probe [18].

Plasmids containing each of the above inserts except pAT-A were cut with appropriate restriction endonucle- ases, and insert fragments were isolated by low-melting- point gel electrophoresis and radiolabeled with 32p_ deoxycytidine triphosphate by the random-primer method [ 19]. Plasmid containing the C4 probe (pAT-A) was ~2p-labeled by nick-translation [20].

Restriction fragment length polymorphism analysis

1. The 5'-end polymorphism of the C2 gene was detected by SstI restriction digest in conjunction with the C2 probe. Five SstI restriction fragment length polymorphisms (RFLPs) of 2.75, 2.7, 2.65, 2.55, and 2.4 kb have been described in the normal population [10, 13].

2. The TaqI restriction digest in conjunction with Bf probe identified two TaqI RFLPs of 6.6 and 4.5 kb at the 3' end of the C2 gene [21].

3. The TaqI polymorphism at the 5' end of the C4 genes described as 7.0, 6.4, 6.0, and 5.4 kb represented the C4A gene, C4A gene deletion, C4B long gene, and C4B short gene, respectively [22].

4. TaqI DNA-RFLP allogenotyping of HLA-DR/DQ was established using the combination of DR/3, DQ~, and DQ,8 probes as previously described [23].

Statistical analysis. Statistical comparison was per- formed using (a) either chi-square 0¢ 2) or chi-square with Yates' correction (X2Va~es,), (b) Fisher's exact test, and (c) the hypothesis t-test wherever appropriate.

RESULTS

Prevalence of thyroid autoantibody. From 217 members of 21 families, 51 and 166 members, respectively, were affected and unaffected with GD. The presence of thyroid autoantibodies (antimicrosomal and antithyroglobu- lin) were found in 45 of 166 unaffected members. The prevalence of thyroid autoantibodies in unaffected members of each family varied from 0 to 67%, with a mean of 27.1% (see Table 1).

Recombination and family extended haplotypes. MHC- extended haplotypes with an approximate genetic distance of 3000 kb (from HLA-A centromeric to the HLA- DR/DQ complex) were constructed in each family by using both phenotypic and genotypic markers as de-


scribed in Materials and Methods. Five recombinations were found in unaffected members in whom thyroid autoantibodies were not detected, three occurred between HLA-A and HLA-B, and two between HLA-B and the MHC class III from 296 and 284 informative meioses, respectively. No recombination was found between the MHC class III and II or between the MHC class II subregions (HLA-DR/DQ complex and HLA- DQA2) in 285 and 160 informative meioses, respectively.

In an attempt to identify a GD-associated haplotype, all family-extended haplotypes were classified into four categories based on their association with either affected or unaffected members, with or without thyroid autoantibodies as follows: (a) haplotypes inherited by members who were affected with GD (regardless of sharing by the unaffected member) were assigned as affected (Aft) haplotypes; (b) Aft haplotypes that were shared by thyroid-autoantibody-positive (Ab + ve)/unaffected members were considered as shared haplotypes and re- classified as Aff/Ab+ve haplotypes; (c) all unaffected haplotypes inherited by Ab+ve/unaffected members (except those shared with Aft members) were classified as Ab+ve haplotypes, regardless of sharing by the thyroid-autoantibody-negative (Ab - ve)/unaffected members; and finally (d) all of the remaining extended haplotypes that were shared between Ab-ve/unaffected members (i.e., not shared by either Affor Ab + ve members) were classified as A b - v e haplotypes. Based on these criteria, a total number of 159 extended haplotypes deduced from 217 members of 21 families of patients with GD were classified as 40 Aft, 31 Aff/Ab + ve, 29 Ab+ve, and 59 A b - v e haplotypes. Complete extended haplotypes in each category including the five recombination haplotypes are shown in Table 2. The frequencies ofpolymorphic alleles from each locus were statistically compared individually or in combinations among the four categories of extended haplotype. Table 3 summarizes polymorphic markers that statistically are significantly different among categories of extended haplotypes. The frequencies of HLA-B8, C4A*QOB* 1, the 6.4-kb TaqI/C4B, and HLA-DR17 (DRIll71 and fl172/ DQ~2/DQ~2a DNA-RFLP) individually or in combination were found significantly increased in the Aft when compared with the A b - v e haplotypes. The frequencies of either HLA-B8 and DR17 alone or in combination were also found to be significantly increased in the Aff/Ab + ve when compared with the Ab - ve haplotype. Though the frequencies of these HLA markers were consistently higher in the Ab + ve when compared with the A b - v e haplotypes, the differences did not reach statistical significance. This might be due to the small number of haplotypes in this category since over 50% of the haplotypes were shared by members affected with

GD and were classified as Aff/Ab + ve haplotypes. When statistical comparisons were repeated after pooling two categories of Ab + ve haplotypes, the significance was very much the same as seen in the comparisons made between the Aff/Ab + ve and Ab - ve haplotypes, except for the loss and gain of the significant increase in the frequencies of the DQ~2 and the 6.4-kb TaqI/C4B, respectively (see Table 3).

The significance of associations of either single or multiple genetic markers from the HLA-B8, C4A*Q0/ 6.4-kb TaqI, HLA-DR17/DQ2 haplotype with the Aft (38 of 51 = 74.5%) and Ab+ve (33 of 45 = 73.3%) when compared with A b - v e (64 of 121 = 52.9%) members were also observed when analysis was per- formed by direct counting of the number of individuals who possess these HLA markers in each subgroup, X 2 = 10.12, df = 2, p < 0.0064 (see Table 4).

Linkage disequilibrium between MHC polymorphisms on Graves' disease family-extended haplotypes. There were no significant differences in terms of two-locus linkage disequilibrium (LD) among the HLA-B, MHC class III (C2, Bf, C4A, and C4B), and HLA-DR/DQ complex in the four categories of extended haplotypes. The association between HLA-B and DR with the 5 '-end-C2 and 3'-end- C2/Bfpolymorphisms from 159 extended haplotypes is shown in Table 5, and the significance of the associations are summarized in Table 6. Both 5 '- and 3 '-end polymorphisms of C2 are in strong LD with certain HLA-B and DR haplotypes. At the 5' end of C2, the 2.7-kb SstI is in LD with the HLA-B8, DR3 haplotype while the 2.65- kb SstI is in LD with the HLA-DR4, DR5 haplotype and the 2.4-kb SstI with the HLA-B44, DR7 haplotype. At the 3' end of C2, HLA-B44, DR7 haplotype also is in LD with the 6.6-kb TaqI and the Bf*F while the B18, DR3 (rather than B8) is in LD with the 4.5-kb TaqI and the Bf*F I (see Table 6).

The protein polymorphisms of C4A and C4B are also strongly correlated with the structural polymorphisms of the C4B gene and in LD with several HLA-DR haplotypes. The correlation and association between C4 phenotype, C4B gene polymorphism, and HLA-DR 3 TaqI-RFLP on 159 extended haplotypes are shown in Table 7, and the frequencies of C4B gene polymorphisms associated with HLA-DR haplotypes defined by the DR/~ TaqI-RFLP are summarized in Table 7. Over 80% of the C4B genes on HLA-DRf1171, DRIll4, DRfl7 and DRIll0 are structurally short genes (6.4- and 5.4-kb TaqI), whereas on the HLA-DRf115;16, DRfl4, DR/311;12, and DR/39 are structurally long genes (6.0- kb TaqI). Associations between HLA-B and C4 polymorphisms were also observed, but.the majority of these associations were not significant after correction for the number of comparisons. Table 8 summarizes the

103

T A B L E 2 A tota l o f 159 M H C - e x t e n d e d h a p l o t y p e s d e d u c e d f r o m 217 m e m b e r s o f 21 fami l i es o f p a t i e n t s

w i t h G r a v e s ' d i s ea se w e r e c lass i f ied in to f o u r c a t e g o r i e s as (a) 40 A f t h a p l o t y p e s , (b) 31 A f f / A b + ve

h a p l o t y p e s , (c) 29 A b + v e h a p l o t y p e s , and (d) 59 A b - v e h a p l o t y p e s ; five r e c o m b i n a t i o n h a p l o t y p e s are also s h o w n at t he e n d o f this t ab le

Class I C2-RFLP C4 prot C4/TaqI-RFLP Class II DNA-RFLP

HLA A B Bw SstI TaqI Bf C4A C4B C4A/21A C4B/21B DR~ DQa DOff DXez Family

a) Affected haplotypes (n = 40) 1) 9 44 4 2.75 4.5 S A3 B2 7.0/3.2 6.0/3.7 11 2 3b U A 2) 3 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 lb lb L A 3) 2 44 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 3 3b L B 4) 2 39 6 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 8 lb 3a L D 5) 3 35 6 2.7 4.5 F A3 A2 7.0/3.2 6.0/3.7 1 la la L E 6) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U E 7) 3 35 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 2 2a U E 8) 3 17 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 11 2 3b L E 9) 2 62 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 4 3 3a L F

10) 29 44 4 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 7 ~ 3 2b L F 11) 3 5 4 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 1 la la L F 12) 3 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 13a I lc lb L G 13) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 2 2a U G 14) 10 14 6 2.7 4.5 S A3 B1 7.0/3.2 5.4/3.7 13a ~ lb la U G 15) 28 44 4 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 71 3 2b L G 16) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U J 17) 11 18 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 172 2 2a L J 18) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U K 19) 2 7 6 2.65 4.5 S A4 B2 7.0/3.2 5.4/3.7 1 la la L K 20) 1 13 4 2.7 4.5 S A3 B1 7.0/3.2 5.4/3.7 72 3 2b L K 21) l l 18 6 2.7 4.5 F I A3 BQ0 7.0/-- - - /3.7 172 2 2a L L 22) 32 7 6 2.65 4.5 S A6 B1 7.0/3.2 5.4/3.7 11 2 3b L L 23) 2 44 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 15 lb lb U L 24) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U M 25) 2 27 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 3 3a U M 26) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U M 27) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 2 2a U N 28) 29 5 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 lb lb U N 29) 28 44 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 lb lb U N 30) 3 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 lb lb U O 31) l 53 4 2.7 4.5 F A3 B1 7.0/3.2 6.0/3.7 13a ~ lb la L O 32) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 j 2 2a U O 33) 3 27 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 16 lb la L P 34) 29 8 6 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 3 3a u P 35) 9 35 6 2.65 4.5 s A3 B1 7.0/3.2 6.0/3.7 11 2 3b L R 36) 11 35 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 2 2a U S 37) 9 37 4 2.7 4.5 F A3 B1 7.0/3.2 5.4/3.7 10 la la U S 38) 29 44 4 2.7 4.5 S A4 B5 7.0/3.2 5.4/3.7 14a la la L T 39) 3 35 6 2.7 4.5 F A3 A2 7.0/3.2 6.0/3.7 1 la la L U 40) 2 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 4 3 3a L U

b) A f f / A b + v e haplotypes (n = 31) 1) 28 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 t 2 2a U A 2) 3 5 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 1 la la L A 3) 28 14 6 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 11 2 3b L A 4) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 2 2a U B 5) 32 44 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 3 3b L B 6) 2 35 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 8 lb 3a L B 7) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U B 8) 28 44 4 2.65 4.5 S AQ0 B2 7.0/3.2 5.4/3.7 13a ~ lb la U C 9) 2 8 6 2.4 6.6 F A3 BQ0 7.0/3.2 6.0/3.7 4 3 3b L C

10) 2 44 4 2.7 4.5 F A3 B1 7.0/3.2 5.4/3.7 7 ~ 3 2b L C 11) 28 44 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 1 la la L C 12) 2 8 6 2.4 6.6 S A3 A3 7.0/3.2 6.0/3.7 171 2 2a U C 13) 11 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 2 2a U C

(continued)

1 0 4

TABLE 2 A total of 159 MHC-extended haplotypes deduced from 217 members of 21 families of patients with Graves' disease were classified into four categories as (a) 40 Aft haplotypes, (b) 31 Aff/Ab + ve haplotypes, (c) 29 Ab +ve haplotypes, and (d) 59 A b - v e haplotypes; five recombination haplotypes are also shown at the end of this table (Continued)

Class l C2-RFLP C4 prot C4/TaqI-RFLP

HLA A B Bw SstI TaqI Bf C4A C4B C4A/21A C4B/21B

Class II DNA-RFLP

DR/3 DQa DOff DXa Family

14) 11 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 15) 3 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 1 16) 1 18 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 14a 17) 28 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 18) 10 41 6 2.7 4.5 F A3 B1 7.0/3.2 5.4/3.7 172 19) 2 5 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 11 20) 1 17 4 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 72 21) 2 35 6 2.65 4.5 S A4 B2 7.0/3.2 5.4/3.7 1 22) 9 22 6 2.7 4.5 S A4 B5 7.0/3.2 5.4/3.7 172 23) 2 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ 24) 2 27 4 2.65 4.5 S A3 B3 7.0/3.2 6.0/3.7 8 25) 1 17 4 2.65 4.5 S A6 B1 7.0/3.2 5.4/3.7 72 26) 29 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 27) 3 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 28) 2 44 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 12 29) 9 39 6 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 17 j 30) 11 35 6 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 31) 2 40 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171

c) A b + v e haplotypes (n = 29) 1) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 l 2) 2 44 4 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 72 3) 9 44 4 2.65 4.5 S A5 B1 7.0/3.2 6.0/3.7 1 4) 1 40 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 5) 10 5 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 15 6) 1 44 4 2.4 6.6 F A3 BQ0 7.0/3.2 6.0/3.7 13a 1 7) 29 44 4 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 7 ~ 8) 29 44 4 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 17 ~ 9) 28 35 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 11

10) 9 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 11) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 ~ I2) 1 8 6 2.65 4.5 S AQ0 B1 - - / - - 6.4/3.7 172 13) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 13a ~ 14) 10 41 6 2.7 4.5 S A4 B5 7.0/3.2 5.4/3.7 4 15) 10 44 4 2.65 4.5 S A3 B2 7.0/3.2 5.4/3.7 15 16) 3 7 6 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 17) 10 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 72 18) 28 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 17 t 19) 3 35 6 2.65 4.5 S A3 BQ0 7.0/-- - - / 3 . 7 1 20) 28 35 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 21) 1 17 4 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 13a 1 22) 9 44 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 4 23) 29 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 24) 1 8 6 2.7 4.5 S AQ0 BI - - / - - 6.4/3.7 17 ~ 25) 2 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 13a ~ 26) 2 14 6 2.7 4.5 S A3 BI 7.0/3.2 5.4/3.7 7 ~ 27) 2 44 4 2.65 4.5 S A3 B3 7.0/3.2 6.0/3.7 172 28) 32 62 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 29) 11 35 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 9

d) A b - v e haplotypes (n = 59) 1) 1 44 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.76 11 2) 2 5 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 11 3) 2 13 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 4 4) 9 41 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 11 5) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 171 6) l l 40 6 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 71

2 2a U D la la L D la la L D 3 3a L E 2 2a L F 2 3b L G 3 3a L H la la L H 2 2a L H 2 2a L I lb 3a U 1 3 3a L l 3 3a L l lb lb U 3 2 3b L Q 2 2a U Q 3 3a U R 2 2a U T

2 2a U A 3 2b U A la la L A 2 2a U A lb lb L A lc lb L B 3 2b L B 2 2a U B 2 3b U B 3 3a U C 2 2a U C 2 2a U C lb lb U C 3 3a U D lb lb U E 3 3a U F 3 2b L G 2 2a U H Ia la L J 3 3a L I lc lb U K 3 3a L K 3 3a U Q 2 2a U R lb la L R 3 2b U R 2 2a U S 3 3a U S 3 3a L T

2 3b L A 2 3b L A 3 3b U A 2 3b L A 2 2a U A 3 2b L B

(continued)

105

T A B L E 2 (Continued)

HLA

Class I C2-RFLP C4 prot C4/TaqI-RFLP

A B Bw Sstl TaqI Bf C4A C4B C4A/21A C4B/21B

Class II DNA-RFLP

DRfl DQa DOff DXa Family

7) 2 17 4 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 8) 19 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 9) 2 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7

10) 2 17 4 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 11) 3 7 6 2.7 4.5 S AQ0 B1 - - / - - 7.0/3.7 12) 32 44 4 2.4 6.6 F A3 B1 7.0/3.2 5.4/3.7 13) 9 27 4 2.7 4.5 F A3 B1 7.0/3.2 6.0/3.7 14) 2 5 4 2.65 4.5 S AQ0 B2 7.0/3.2 5.4/3.7 15) 2 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 16) 2 40 6 2.65 4.5 S AQ0 B2 7.0/3.2 5.4/3.7 17) 29 35 6 2.65 4.5 S A4 B2 7.0/3.2 5.4/3.7 18) 2 7 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 19) 3 37 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 20) 10 27 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 21) 2 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 22) l 17 4 2.65 4.5 S A3 B1 7.0/3.2 5.4/3.7 23) 28 37 4 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 24) 28 40 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 25) 9 38 4 2.65 4.5 S A2 B1 7.0/3.2 6.0/3.7 26) 10 5 4 2.65 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 27) 2 7 6 2.7 4.5 S A3 BQ0 7.0/3.2 6.0/3.7 28) 32 40 6 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 29) 9 18 6 2.7 4.5 F~ A3 B3 7.0/3.2 6.0/3.7 30) 32 14 6 2.7 4.5 S A3 B1 7.0/3.2 5.4/3.7 31) 3 35 6 2.7 4.5 S A2 BQ0 7.0/3.2 6.0/3.7 32) 3 47 4 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 33) 2 27 4 2.7 4.5 S A3 BQ0 7.0/3.2 5.4/3.7 34) 1 5 4 2.65 4.5 S A3A2 B1 7.0/3.2 6.0/3.7x2 35) 9 7 6 2.7 4.5 S A3 Bl 7.0/3.2 6.0/3.7 36) 29 40 6 2.65 4.5 S A3 BI 7.0/3.2 6.0/3.7 37) 1 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 38) 2 27 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 39) 1 37 4 2.7 4.5 F A3 B1 7.0/3.2 5.4/3.7 40) 2 40 6 2.65 4.5 S A3 BQ0 7.0/-- - - /3 .8 41) 3 18 6 2.7 4.5 F t A3 BQ0 7.0/-- - - /3 .7 42) 29 44 4 2.7 6.6 F A3 B1 7.0/3.2 5.4/3.7 43) 2 44 4 2.4 6.6 F A3 BQ0 7.0/3.2 6.0/3.7 44) 2 62 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 45) 9 7 6 2.7 4.5 S A3A2 BI 7.0/3.2 6.0/3.7x2 46) 2 5 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 47) 2 62 6 2.65 4.5 S A3 B3 7.0/3.2 6.0/3.7 48) 9 44 4 2.7 4.5 S A4 B2 7.0/3.2 5.413.7 49) 29 62 6 2.7 4.5 S A4 B2 7.0/3.2 5.4/3.7 50) 2 5 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 51) 11 22 6 2.7 4.5 F AQ0 B1 7.0/3.2 5.4/3.7 52) 3 7 6 2.7 4.5 S A3 B1 7.0/3.2 6.0/3.7 53) i I 22 6 2.7 4.5 S A4 B5 7.0/3.2 5.4/3.7 54) 29 44 4 2.7 4.5 F A3 BQ0 7.0/3.2 6.0/3.7 55) 9 13 4 2.7 4.5 S A2 BQ0 7.0/-- - - /3 .7 56) 1 44 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 57) 3 7 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 58) 29 44 4 2.7 4.5 F A3 B1 7.0/3.2 5.4/3.7 59) 3 39 6 2.4 4.5 S A4 B2 7.0/3.2 5.4/3.7

R e c o m b i n a t i o n h a p l o t y p e s (n = 5) 1) 29 8 6 2.7 4.5 S AQ0 B1 - - / - - 6.4/3.7 2) 19 8 6 2.7 4.5 S A6 B1 7.0/3.2 5.4/3.7 3) 2 44 4 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 4) 11 62 6 2.65 4.5 S A3 B1 7.0/3.2 6.0/3.7 5) 9 44 4 2.7 4.5 F A3 BQ0 7.0/3.2 6.0/3.7

72 3 3a L B 171 2 2a U B 171 2 2a U B 72 3 3a L B

15 lb lb U C 71 3 2b L C

15 lc lb U C 13a ~ lb la U C 15 lb lb U D 15 lb lb U D

1 la la L D 17 ~ 2 2a U E 15 lb lb U E 4 3 3a L E 4 3 3a L E

171 2 2a U H 15 lb lb U H 4 3 3b L H

13a I lc lb L l 4 3 3a L I

15 lb lb L I 14a la la L J 172 2 2a L J 71 3 2b U J 1 la la L K

Br 2 3b L L 14a la la L L 15 lc x U M 15 lb lb U N 4 3 3a U N

17 l 2 2a U N 15 lb lb U O 10 la la U O 8 lb 3a L O

172 2 2a L P 71 3 2b L P

13a I lc lb L P

4 3 3a U P 15 lb lb U Q 13a ~ lb la L R 4 3 3a U R 1 la la L R

14a la la L R 11 2 3b L S 4 3 3a U S

15 lb lb U T 4 3 3a U T 1 la la L T 72 3 2b L T

11 2 3b L U 13a 4 lc lb U U 71 3 2b L U 8 lb 3a L U

171 2 2a U B 72 3 3a L B 8 lb 3a L B 4 3 3a U S 1 la la L T


T A B L E 3 F requenc ies o f single o r mul t ip le gene t ic marke r s on e x t e n d e d hap lo types o f Graves ' d isease families that are stat ist ically significantly d i f ferent be tween (a) the A f t and A b - ve hap lo types , (b) the A f f / A b + ve and A b - v e haplo types , (c) the A b + ve and A b - v e hap lo types , and (d) all A b + ve ( combined A f f / A b + ve with A b + ve) and A b - ve hap lo types

Genetic markers

Aft Aff/Ab + ve Ab + ve Ab - ve haplotype haplotype haplotype haplotype (n = 40) (n = 31) in = 29) (n = 59)

Fisher's p

(a) ~b) (c) (d)

HLA-B8 0.2500 (10) 0.2581 (8) 0.2069 (6) 0.0678

C4A*QOB*I 0.3000 (12) 0.2258 (7) 0.2414 (7) 0.1186 6.4-kb TaqI/C4B 0.3077 (12) 0.2187 (7) 0.2500 (7) 0.0847

DR/~17*/DQ~2/DQ,82a 0.2500 (10) 0.2903 (9) 0.2069 (6) 0.1017 HLA-DRfi17 ~ 0.2500 (10) 0.2903 (9) 0.2069 (6) 0.1017 HLA-DQc~2 0.4000 (16) 0.4516 (14) 0.3103 (9) 0.2373 HLA-DQfl2a 0.3000 (12) 0.3548 (11) 0.2759 (8) 0.1356 DR17(13171 and/3172) 0.3000 (12) 0.3548 (11) 0.2759 (8) 0.1356 HLA-B8/6.4-kb TaqI 0.2250 (9) 0.1935 (6) 0.2069 (6) 0.0678 HLA-B8/DR17/DQ2 0.2000 (8) 0.2258 (7) 0.1724(5) 0.0678 6.4 kb TaqI/DR17/DQ2 0.2500 (10) 0.2258 (7) 0.2069 (6) 0.0847 Extended haplotype* 0.2000 (8) 0.1935 (6) 0.1379 (4) 0.0678

(4) <0.013 <0.016 NS <0.011

(7) <0.025 NS NS NS (5) <0.007 NS NS <0.024

(6) <0.047 <0.026 NS <0.029 (6) <0.047 <0.026 NS <0.029

(14) NS <0.034 NS NS (8) <0.042 <0.018 NS <0.016 (8) <0.042 <0.018 NS <0.016 (4) <0.026 NS NS <0.032 (4) <0.05 <0.036 NS <0.032 (5) <0.026 NS NS <0.039 (4) <0.05 NS NS NS

The number of haplotypes are shown in parentheses. NS, not significant. *HLA-B8, 2.7-kb SstI-4.5-kb TaqI/C2 Bf*S, C4A*QOB*I-6.4-kb TaqI/C4B, DR17/DQ2 extended haplotype.

significance o f associat ions be tween the C4 pheno type , C 4 B / T a q I - R F L P with H L A - B , and DRf i T a q I - R F L P on 159 e x t e n d e d hap lo types f rom families o f pat ients with G D .

with bo th 5' and 3' p o l y m o r p h i s m s o f the C2 gene on cer ta in haplo types . As shown in Tables 5 and 6, the 2.7- kb Ss t I / 5 ' -C2 is significantly associated with H L A - B 8 , DR3 whereas the the 2 .65-kb SstI is significantly associ- a ted with H L A - D R 4 and DR5. T h e less c o m m o n 2.4-

D I S C U S S I O N

Recombination and linkage disequilibrium in the human MHC. Extens ive inves t iga t ion o f M H C - e x t e n d e d haplo types in 21 families o f pa t ien ts with G D gives fur ther suppor t for LD among the M H C class I, I II , and II regions. Based on the f requenc ies o f r ecombina t ion that occur red at 1% ( three o f 296) be tween H L A - A and B and 0 .7% Itwo o f 284) b e t w e e n H L A - B and the M H C class I I I in this s tudy, the es t ima ted dis tances would be 1.0 and 0.7 cen t i -Morgan , i.e., 1000 and 700 kb be tween H L A - A and B and b e t w e e n H L A - B and class III , respectively. This resul t co r r e sponds with the prec ise dis tance o f 1300 and 650 kb be tween H L A - A and B, and H L A - B and the M H C class I I I , which has recen t ly been estab- l ished [4]. T h e dis tance be tween the M H C class I I I and II is app rox ima te ly 350 kb [24], whereas that be tween the H L A - D R / D Q complex and H L A - D Q A 2 is only 100 kb [25]. Obvious ly , it will r equ i re larger numbers o f families (over 300 and 1000 in format ive meioses , re- spect ively) to es tabl ish any r ecombina t i on that might occur, assuming that there is no "ho t spot" o f r ecombina- t ion in these par t icu lar regions. T h e results o f our study agree with the assumpt ion since no r ecombina t i on was found be tween these regions [26]. In addi t ion , our s tudy also d e m o n s t r a t e d a s t rong LD be tween H L A - B and DR,

TABLE 4 Dis t r i bu t ion o f pos i t ive H L A marke r s (e i ther H L A - B 8 , C 4 A * Q 0 / 6 . 4 - k b TaqI , DR3 , or in combina t ion) in re la t ion to age and sex o f the affected and unaffec ted m e m b e r s with and wi thout thy ro id au toan t ibod ies

HLA Aft Ab + ve Ab - ve markers member member member

Positive Male 8 12 34 Female 30 21 30 Total 38 (74.5) 33 (73.3) 64 (52.9) Mean age 49.8 - 16.3 44.3 + 16.8 b 41.0 -+ 18.8

Negative Male 0 4 30 Female 13 8 27 Total 13 (25.6) 12 (26.7) 57 (47.1) Mean age 49.9 -~ 11.6 54.3 +- 15.1 ~b 43.7 +- 17.8

Both Total 51 45 121 Mean age 49.9 --- 15.1 c 47.0 -+ 16.8 42.3 -+ 18.4 a'~

Percentages of distribution are shown in parentheses and the difference in the number of individuals with positive and negative HLA markers in the three subgroups is statistically significant 0( ~ = 10.12, df = 2, p < 0.0064). The differences in the age (mean -+ SD) are statistically significant with ap < 0.012, bp < 0.04, and ~p < 0.006.


TABLE 5 Association among HLA-B, HLA-DR, and the 5'-end C2/SstI and 3'-end C2/TaqI-RFLP with factor-B (Bf) polymorphisms on 159 extended haplotypes from families of patients with Graves' disease

S s t I / 5 ' - e n d C 2 - R F L P ( k b ) T a q I / Y - e n d C 2 - R F L P ( k b ) a n d B f

H L A 2 . 7 5 2 . 7 0 2 . 6 5 2 . 4 0 T o t a l 4 . 5 ~ S 4 . 5 " F 4 . 5 " F I 6 . 6 " S 6 . 6 " F

B 5 - - 3 8 - - 11 11 . . . .

B 7 - - 12 4 - - I 6 1 6 . . . .

B 8 - - 2 4 2 2 2 8 2 6 - - - - 1 1

B 1 3 - - 3 - - - - 3 3 . . . .

B I 4 - - 4 - - - - 4 4 . . . .

B 1 6 - - 2 1 1 4 4 . . . .

B 1 7 - - 4 3 - - 7 7 . . . .

B 1 8 - - 4 l - - 5 2 - - 3 - - - -

B 2 2 - - 3 - - - - 3 2 1 - - - - - -

B 2 7 - - 3 4 - - 7 6 1 - - - - - -

B 3 5 - - 5 9 - - 1 4 12 2 - - - - - -

B 3 7 - - 4 - - - - 4 2 2 - - - - - -

B 4 0 - - 3 1 l 1 15 1 4 - - - - - - 1

B 4 1 - - 2 1 - - 3 2 1 - - - - - -

B 4 4 1 8 12 7 2 8 1 7 3 - - - - 8

B 4 7 - - 1 - - - - 1 l . . . .

B 5 3 - - 1 - - - - 1 - - 1 - - - - - -

B 6 2 - - 2 3 - - 5 5 . . . .

T o t a l 1 8 8 5 9 11 1 5 9 1 3 4 11 3 1 1 0

0 . 6 c~ 5 5 . 3 c x 3 7 . 1 % 6 . 9 % 8 4 . 3 % 6 . 9 ~ 1 . 9 % 0 . 6 % 6 3 ° ~

D R 1 - - 8 7 - - 15 12 3 - - - - - -

D R 2 - - 1 6 5 - - 2 1 2 0 1 - - - - - -

D R 3 - - 3 3 4 2 3 9 3 3 1 3 1 1

D R 4 - - 6 2 0 1 2 7 2 5 1 - - - - 1

D R 5 1 1 11 - - 13 1 3 . . . .

D R 6 - - 1 0 6 2 1 8 15 1 - - - - 2

D R 7 - - 11 2 5 1 8 1 0 2 - - - - 6

D R 8 - - 1 3 1 5 5 . . . .

D R 9 - - - - 1 - - 1 1 . . . .

D R 1 0 - - 2 - - - - 2 - - 2 - - - - - -

T o t a l 1 8 8 5 9 11 1 5 9 1 3 4 11 3 1 1 0

0 . 6 % 5 5 . 3 % 3 7 . 1 % 6 . 9 % 8 4 . 3 % 6 . 9 % 1 . 9 ~ 0 . 6 % 6 . 3 %

kb SstI/5'-C2 and the 6.6-kb TaqI/3'-C2 and the Bf*F alleles are all associated with HLA-B44, DR7 as part of the extended haplotype. Interestingly, the TaqI structural polymorphism of the C4B gene has a strong correlation with C4 phenotypes and is significantly associated with certain HLA-DR/3 TaqI-RFLP. As shown in Table 7, all of the C4B'5 and C4B'2 genes (except one) are structurally short (5.4 kb) whereas all C4B'3 genes are structurally long (6.0 kb). The sizes of C4B'1 genes depend on the association with the C4A phenotype and HLA-DR/3 TaqI-RFLP. All C4B'1 genes following the C4A'6, C4A*Q0 (except one) or in association with HLA-DR/317 i, DR37, and DR/310 are structurally short (either 5.4- or 6.4-kb TaqI) whereas C4B'1 genes following the C4A'2, C4A'5 or in association with HLA-

DR/~I;Br, DR/~15;16, DRI34, DRB11;12, DRB13;14, DR/~8, and DR/39 are structurally long (6.0 kb).

Autoimmune thyroid disease-associated HLA markers and haplotype. The attempt to identify a genetic marker that is particularly associated with Aft members of multiplex families with GD was unsuccessful with the ten markers (four phenotypic and six genotypic markers spanning from HLA-A to the HLA-DR/DQ complex) used in this study. Several genetic markers on the HLA-B8, C4A*Q0/6.4-kb TaqI, DR17/DQ2 haplotype, were significantly associated with both Aft and unaffected members in whom thyroid autoantibodies were positive as compared with the Ab-re/unaffected members of the families, suggesting a common genetic background

1 0 8

TABLE 6 Significance of associations among HLA-B, HLA-DR, and the C2 DNA-RFLP and factor-B (Bf) protein polymorphism on 159 extended haplotypes from families of patients with Graves' disease

X 2

C 2 - R F L P N u m b e r o f (O - E) 2

H L A - B and Bf Fisher 's

a n d D R p o l y m o r p h i s m (kb) p d f Pc 0 E E

5 'C2 /Ss t I

B8 2.70 < 2 . 4 x 10 -4 17 x 3 < 0 . 0 1 2 24 15.5 4.7 < 0 . 0 2 9

B5 2.65 < 0 . 0 1 5 17 x 3 NS 8 4.1 3.7 N S

B35 2.65 < 0 . 0 3 17 x 3 NS 9 5.2 2.8 N S

B40 2.65 < 3 . 3 x 10 -~ 17 x 3 NS 11 5.6 5.2 < 0 . 0 2 2

B44 2.40 < 5 . 3 x 10 -4 17 x 3 < 0 . 0 2 7 7 1.9 13. 7 < 5 x 10 -4

DR2 2.70 < 0 . 0 3 2 9 x 3 NS 16 11.6 1.7 NS

DR3 2.70 < 1 . 4 x 10 -5 9 x 3 < 4 × 10 -4 33 21.6 6.0 < 0 . 0 1 4

D R 4 2.65 < 2 . 2 x 10 -5 9 x 3 < 6 x 10 -4 20 10.0 10.0 < 0 . 0 0 2 1

DR5 2.65 < 3 . 8 × 10 -4 9 × 3 < 0 . 0 2 11 4.8 8.0 < 0 . 0 0 5 1

DR7 2.40 < 3 . 3 x 10 -~ 9 x 3 NS 5 1.2 12.0 < 0 . 0 0 0 9

3 ' C 2 / T a q I Bf

B18 4 .5"F < 1 . 6 x 10 -5 17 × 4 < 0 . 0 0 2 3 0.1 84.1 < 2 × 10 -8

B44 6.6"F~ < 1 . 3 × 10 -5 17 x 4 < 0 . 0 0 1 8 1.8 21.4 < 5 x 10 -5

D R 3 4.5~F < 0 . 0 1 4 9 x 4 NS 3 0.7 7.6 < 0 . 0 0 6 1

DR1 4.5"F~ < 0 . 0 3 9 9 x 4 NS 3 1.0 4.0 < 0 . 0 4 3

D R 1 0 4 .5"F < 4 . 4 x 10 -~ 9 x 4 N S 2 0.1 36.1 < 3 × 10 -6

D R 7 6 .6"F < 1 . 5 × 10 -4 9 × 4 < 0 . 0 0 6 6 1.1 21.8 < 5 × 10 -~

df, deg rees of f r eedom; O, obse rved haplotype; E, expec ted haplo type (calculated f rom f requencies o f the two m a r k e r s in the popu la t ion study); a n d N S , n o t s i g n i f i c a n t .

TABLE 7 Association among C4 phenotypes, C4B TaqI polymorphism, and HLA-DR/~ DNA-RFLP on 159 extended haplotypes obtained from families of patients with Graves' disease

C4B HLA-DR/~ D N A - R F L P C4 T a q I

p h e n o t y p e (kb) Tota l 1,Br 15,16 171 172 4 11,12 13 14 7 8 9 10

A*QOB*I 7.0 1 - - 1 . . . . . . . . . .

6.4 31 - - - - 27 1 2 - - 1 . . . . .

5.4 1 . . . . 1 . . . . . . .

A*QOB*2 5.4 3 - - 1 . . . . 2 . . . . .

A * 2 B * Q 0 6.0 1 1 . . . . . . . . . . .

De l e t i on 1 . . . . . . . . 1 - - - - - -

A*2B*I 6.0 1 . . . . . . 1 . . . . .

A*3B*Q0 6.0 14 1 2 - - - - 8 1 2 . . . . .

5.4 1 . . . . . . . 1 . . . .

De l e t i on 4 1 - - - - 2 . . . . . 1 - - - -

A*3B*I 6.0 49 3 14 - - 1 13 9 6 1 - - 1 1 - -

5.4 17 - - - - 2 1 . . . . 12 - - - - 2 A*3B*2 6.0 1 . . . . . 1 . . . . . .

5.4 1 - - 1 . . . . . . . . . .

A*3B*3 6.0 4 - - - - - - 2 1 . . . . 1 - - - - A*3A*2 6.0 2 2 . . . . . . . . . . .

A*3A*2B* I 6.0 2 - - 2 . . . . . . . . . .

A*3A*3 6.0 1 - - - - 1 . . . . . . . . .

A*4B*2 5.4 11 6 - - 1 . . . . 2 - - 2 - - - -

A*4B*5 5.4 4 - - - - - - 1 2 - - - - 1 . . . . A*5B*I 6.0 1 1 . . . . . . . . . . .

A*6B*I 5.4 8 . . . . . 2 1 - - 5 - - - - - -

Tota l 159 15 21 31 8 27 13 13 5 18 5 1 2


TABLE 8 Significance of associations among HLA-B, HLA-DR, and the C4 protein and TaqI/DNA-RFLP on 159 extended haplotypes from families of patients with Graves' disease

X 2

Number of (O -- E) 2 HLA-B and C4 protein Fisher's

DR TaqI-RFLP p df Pc 0 E E p

B8 A*QOB*I <3 x 10 ~' 14 x 18 <0.0006 25 5.8 63.6 <1 × 10 -7 DR/3171 A*QOB*I <2 x 10 ~ 14 x 11 <0.0003 27 6.4 66.3 <8 × 10 -8 DR/313 A*QOB*2 <0.018 14 x 11 NS 2 0.2 16.2 <0.0003 DR,84 A*3B*Q0 <4 × 10 4 14 x 11 NS 8 3.2 7.2 <0.0075 DR/3172 A*3B*3 <0.013 14 × 11 NS 2 0.2 16.2 <0.0003 B44 A*3B*2 <0.031 14 × 18 NS 2 0.4 7.8 <0.0056 B39 A*4B*2 <3 × 10 4 14 × 18 NS 3 0.2 37.1 <2 × 10 ~' DR/~I,Br A*4B*2 <9 × 10 -5 14 x 11 <0.014 6 1.0 25.0 <2 × 10 5 DR/314 A*4B*2 <0.039 14 x 11 NS 2 0.3 9.6 <0.0025 DR38 A*4B*2 <0.039 14 × 11 NS 2 0.3 9.6 <0.0025 B22 A*4B*5 <0.0015 14 × 18 NS 2 0.1 46.1 <7 × 10 7 B17 A*6B*I <9 × 10 -5 14 × 18 <0.0023 4 0.4 38.1 <2 × 10 (' DR~7 A*6B*I <5 x 10 -4 14 × 11 NS 5 0.9 18.7 <0.0001 DR/31,Br A*3A*2 <9 x 10 ~ 14 x 11 NS 2 0.2 16.2 <0.0003 DR/315,16 A*3A*2B*I <0.017 14 x 11 NS 2 0.3 9.6 <0.0025

B8 6 .4kb < 7 × 10-" 4 × 18 < 5 × 10 -5 25 5.5 69.1 < 6 × 10 -s DR/317 t 6 .4kb <7 × 10-" 4 × 11 <4 × 10 -5 31 6.0 104.2 <5 × 10 -9 B5 6.0 kb <0.022 4 × 18 NS 9 5.3 2.6 NS B27 6.0 kb <0.049 4 × 18 NS 6 3.4 2.0 NS B35 6 .0kb <0.018 4 × 18 NS 11 6.8 2.6 NS DR/315,16 6.0 kb <0.0015 4 × 11 NS 18 10.2 6.0 <0.014 DR/34 6.0 kb <2 × 10 .4 4 × 11 <0.006 22 13.1 6.0 <0.014 DR/311,12 6 .0kb <0.0064 4 × 11 NS 11 6.3 3.5 NS B14 5.4 kb <0.0064 4 × 18 NS 4 1.2 7.0 <0.0085 B22 5.4 kb <0.024 4 × 18 NS 3 0.9 5.2 <0.022 B39 5 .4kb <0.024 4 × 18 NS 3 0.9 5.2 <0.022 DR/314 5 .4kb <0.025 4 x 11 NS 4 1.4 4.8 <0.027 DR/37 5.4 kb <6 x 10 -~ 4 × 11 <3 × 10 -5 17 5.2 26.8 <2 × 10 -5 B18 Deletion <0.0077 4 × 18 NS 2 0.2 21.2 <5 × 10 -5 DR/3172 Deletion <0.021 4 × 11 NS 2 0.3 9.6 <0.0025

df, degrees of freedom; O, observed haplotype; E, expected haplotype (calculated from frequencies of the two markers in the population study); and NS, not significant.

across the spectrum of autoimmune thyroid disease. Thyroid autoantibodies were found to be present in 6%-9% of the general population [3, 27] (and our unpublished data), but its HLA association has never been reported. Thyroid autoantibody has been shown to be a good marker for symptomless autoimmune thyroiditis. Thyroid functional abnormalities on thyroid-releas- ing hormone testing were found in 26% of symptomless Ab + ve individuals, whereas only 2.8% of A b - ve sub- jects showed abnormal response [28]. Thyroid microsomal antibody activity was also found to be a good marker for the development of postpartum thyroiditis in pregnancy [29-31]. The prevalence of thyroid autoantibodies in unaffected members of families in our study was significantly higher than in the general population (27.1% vs 9%, p < 0.001). This could be a direct effect of having a higher frequency of HLA-B8, C4A*Q0/6.4-kb TaqI, DR3 in these families. A prepon-

derance of females over males affected with GD was observed with the ratio of 5.4 (43 : 8); however, the ratio was only 1.8 (29:16) for Ab+ve. The overall female-male ratio in these families was 1.5 (129 : 88). This sexual preponderance agrees with the prevalence of autoimmune thyroid disease in the general population [3, 27]. The prevalence of thyroid autoantibody increases with age, being detected in 13%-15% of an elderly population (over 70 years old) with a mean age of 76.2 -+ 5.0 [32]. The possibility of increased prevalence of thyroid autoantibodies later in life in the A b - v e members cannot be excluded since the mean age of this subgroup was significantly lower than that of the Ab + ve with HLA marker-ve subgroup (p < 0.012). Interest- ingly, the significant difference in the mean age of family members with positive and negative HLA markers within the Ab+ve subgroup (p < 0.04) suggests that the production of thyroid autoantibody occurred earlier


in those who were positive than those who were negative for HLA markers.

The etiology of autoimmune disease is complex and likely to be multifactorial in origin. Though the increased prevalence of autoimmune disorders in families of patients with autoimmune disease suggests genetic susceptibility, the inheritance of disease-susceptible candidate genes (i.e., HLA-DR17/DQ2) is not always associated with the disease. In type-I diabetes (IDDM), the concor- dance of the disease in monozygotic twins is 34% and the disease itself usually occurs within 6 years of the diagnosis being made in the first twin (range, 0 .1 -6 years with a median period of 1 year), with the mean age of diagnosis being 18 years (range, 5 -44 years). Two-thirds of twins remain discordant for between 2 and 24.5 years of follow-up, with the median follow-up being 9 years [33]. Unlike IDDM, GD can develop in awide age range over a long period of time and is not confined to the areas of temperate climate. A pair of genetically identical twins in one of the families in this study developed GD 21 years apart in different parts of the world (temperate and tropical climate), with the age of onset of the disease at 28 and 49 years, respectively.

HLA antigens are in LD, with the strongest LD ap- pearing to be within the HLA-DR/DQ complex [23, 34]. This could be the explanation for the unsuccessful attempts to identify an isolated genetic marker for disease susceptibility. Alternatively, this could suggest that there is no such single disease-specific susceptibility locus for autoimmune disease, but rather that the combination of polymorphic loci on the extended haplotype pre- disposes individuals to autoimmune disease. Further investigation of these families within the HLA-DP locus that is not in strong LD with HLA-DR/DQ complex and the rest of the haplotype supports this latter explanation (S. Ratanachaiyavong and A.M. McGregor, manuscript in preparation). The inheritance pattern of autoimmune thyroid disease in these families is quite different and does not conform to a simple straightforward genetic model. We are in the process of seeking to find the best- fit model for the genetic analysis of these families.

In conclusion, the results of our study suggest that (a) certain HLA class II and III polymorphisms are in strong LD, particularly the disease-susceptibility haplotypes; and (b) while either single or multiple combinations of genetic markers on the HLA-B8, 2.7-kb SstI/4.5-kb TaqI Bf*S, C4A*Q0/6.4-kb TaqI C4B'1 , HLA-DR17/ DQ2 extended haplotype are positively associated with autoimmune thyroid disease, only GD is significantly associated with this extended haplotype.

ACKNOWLEDGMENTS

We thank Dr J.L. Bidwell, UK Transplant Service, Bristol, Dr D. Larhammar, Department of Cell Research, the Wallenberg

Laboratory, Uppsala, Sweden, and Dr R.D. Campbell, MRC Immunochemistry Unit, Department of Biochemistry, Ox- ford, UK, who kindly provided the MHC class II and class IlI region probes for our study.

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mhc-extended haplotypes in families of patients with grave's disease

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