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CIiniral Endocrinology (1988), 29, 539-547 HLA CLASS I1 DNA GENOTYPES IN GRAVES’ DISEASE: CLUES TO INHERITANCE OF THE HLA-LINKED COMPONENT OF SUSCEPTIBILITY J. FLETCHER, J. A. FRANKLYN, S. M. McLACHLAN, E. YOUNG and M. C. SHEPPARD Department of Medicine. Birmingham University, and Department of Pathology, University of Newcastle upon Tyne, UK (Received25 March 1988; rerurnedfor revision 2 June 1988:/inaily reuised 16 June 1988: accepted6 July 1988) SUMMARY Restriction fragment length polymorphism analysis using DQa. DQP and DRB cDNA probes was performed in Graves’ disease patients and control subjects. The following restriction fragment patterns were increased in frequency in patients compared with control subjects: IO+7.0+4.0kb DRfiITaqI fragments (66% us 32%; Pc0.01; corrected P<0.06), 7.0+4.0kb DQPIBamHI fragments (55% us 15%; P<O.OOI; corrected Pc0.006). and a DQa/TaqI 4.6kb fragment (75% us 36%; PcO-005; corrected P<0-02). These associations could be accounted for by the known association of the B8-DR3 haplotype with the disorder. No non-DR3-related restriction fragment pattern was associated with the disease using any of the probes with restriction enzymes TaqI and BamHI. The lO+7.0+4.0kb DRPITaqI restriction pattern was identified in 23 of 35 Graves’ disease patients. All 23 subjects were heterozygous for this pattern. This was inconsistent with simple recessive inheritance of the DR3-associated component of disease susceptibility (P= 0.01). The implications of these findings are discussed with reference to models for the inheritance of the HLA-linked component of Graves’ disease susceptibility. The aetiology of Graves’ disease is unknown but a role for autoimmune mechanisms is supported by evidence implicating the HLA system in disease predisposition. In white Caucasoid populations an association exists with HLA-B8 and HLA-DR3 (Farid et al., 1979; Allannic et al., 1980). The aetiological fraction for DR3 has been shown to be higher than that for B8 (Stenszky et af., 1985)and other published studies have also found that DR3 is present in a higher proportion of Graves’ disease patients than is the case for B8 (Tiwari & Terasaki, 1985). This suggests that the HLA-linked Graves’ disease susceptibility gene maps closer to HLA-DR than HLA-B. Associations of Graves’ disease with HLA class I (A, B and C) Correspondence: Dr J. Fletcher, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK 539

HLA CLASS II DNA GENOTYPES IN GRAVES' DISEASE: CLUES TO INHERITANCE OF THE HLA-LINKED COMPONENT OF SUSCEPTIBILITY

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Page 1: HLA CLASS II DNA GENOTYPES IN GRAVES' DISEASE: CLUES TO INHERITANCE OF THE HLA-LINKED COMPONENT OF SUSCEPTIBILITY

CIiniral Endocrinology (1988), 29, 539-547

HLA CLASS I1 DNA GENOTYPES IN GRAVES’ DISEASE: CLUES TO INHERITANCE OF THE

HLA-LINKED COMPONENT OF SUSCEPTIBILITY

J . FLETCHER, J . A. FRANKLYN, S. M. McLACHLAN, E. YOUNG and M . C. SHEPPARD

Department of Medicine. Birmingham University, and Department of Pathology, University of Newcastle upon Tyne, UK

(Received25 March 1988; rerurnedfor revision 2 June 1988:/inaily reuised 16 June 1988: accepted6 July 1988)

S U M M A R Y

Restriction fragment length polymorphism analysis using DQa. DQP and DRB cDNA probes was performed in Graves’ disease patients and control subjects. The following restriction fragment patterns were increased in frequency in patients compared with control subjects: IO+7.0+4.0kb DRfiITaqI fragments (66% us 32%; P c 0 . 0 1 ; corrected P<0.06), 7.0+4.0kb DQPIBamHI fragments (55% us 15%; P<O.OOI; corrected Pc0.006). and a DQa/TaqI 4.6kb fragment (75% us 36%; PcO-005; corrected P<0-02). These associations could be accounted for by the known association of the B8-DR3 haplotype with the disorder. No non-DR3-related restriction fragment pattern was associated with the disease using any of the probes with restriction enzymes TaqI and BamHI.

The lO+7.0+4.0kb DRPITaqI restriction pattern was identified in 23 of 35 Graves’ disease patients. All 23 subjects were heterozygous for this pattern. This was inconsistent with simple recessive inheritance of the DR3-associated component of disease susceptibility ( P = 0.01).

The implications of these findings are discussed with reference to models for the inheritance of the HLA-linked component of Graves’ disease susceptibility.

The aetiology of Graves’ disease is unknown but a role for autoimmune mechanisms is supported by evidence implicating the HLA system in disease predisposition. In white Caucasoid populations an association exists with HLA-B8 and HLA-DR3 (Farid et al., 1979; Allannic et al., 1980).

The aetiological fraction for DR3 has been shown to be higher than that for B8 (Stenszky et af . , 1985) and other published studies have also found that DR3 is present in a higher proportion of Graves’ disease patients than is the case for B8 (Tiwari & Terasaki, 1985). This suggests that the HLA-linked Graves’ disease susceptibility gene maps closer to HLA-DR than HLA-B. Associations of Graves’ disease with HLA class I (A, B and C)

Correspondence: Dr J. Fletcher, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK

539

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540 J . Fletcher et al.

antigens would thus be accounted for by linkage disequilibrium with class I1 (HLA-D) markers (Baur et al., 1984). The high frequency of DR3 in the control (healthy) population, suggests, however, that this antigen may not correspond directly with the primary disease susceptibility gene, although the involvement of other gene(s) and environmental factors is probably also relevant in this context.

Previous studies of HLA and Graves' disease have mainly employed cellular and serological typing methods. These techniques are limited by the range of typing cells and antisera available. Restriction fragment length polymorphism (RFLP) analysis using gene probes corresponding to the component a and j.? chain genes of the class I1 HLA region allows the direct analysis of variability in this region at the DNA level. This may permit closer associations with Graves' disease to be defined. It may also reveal HLA associations in the substantial proportion (30-50%) of Graves' patients who do not express an associated HLA antigen. In this study, we present the results of an analysis of class I1 HLA DNA polymorphisms in Graves' disease. The implications of our findings for the mode of inheritance of Graves' disease are discussed.

METHODS

Patients and normal controls

Thirty-three unrelated, healthy control subjects (Birmingham) with no personal or family history of autoimmune thyroid disease were randomly selected from laboratory and medical staff and blood donors. These subjects were not DR-typed. Thirty-six Graves' disease patients (Birmingham) were serially recruited for study on the basis of past or present raised serum total T3 and/or total T4, plus the presence of microsomal antibodies, with a diffuse uptake pattern on the wmTc scan, and/or ophthalmopathy. These subjects were not DR-typed.

In addition, 32 healthy control subjects (Birmingham) and 21 Graves' disease patients (Newcastle) were selected for the presence of the DR3 antigen from panels of previously DR-typed subjects. The clinical features of the DR-typed Graves' disease patients were similar to those for non-DR-typed patients, but they were additionally all positive for TSH receptor antibodies at presentation, and all had diffuse uptake patterns on ""Tc scan. DR-typed control subjects had been recruited using the same criteria as non-DR- typed subjects.

All subjects were of white Caucasoid racial origin.

Serological HLA-DR typing and RFLP analysis

For the RFLP studies, DNA was isolated from peripheral blood by the method of Sykes (1983). Ten micrograms of DNA were digested with 30 units of the appropriate restriction enzyme (BamHI or TaqI (BRL, Glasgow, UK)). Conditions for restriction digestion were: BamHI, 37"C, overnight; TaqI, 65"C, 4-6 h. Digested DNA was separated by electrophoresis in 0.7% agarose at 50V, 25 mA for 18-22 h and blotted by the method of Southern onto nylon filters (Hybond-N, Amersham International) (Southern, 1975).

Three cDNA probes were used: (i) the AvaI insert of pII-B- 1 (DQB chain) (Larhammar er al., 1982); (ii) the 500-base-pair PstI fragment of pII-fl-4 (corresponding to the second domain, transmembrane, cytoplasmic and 3'-untranslated portions of DRB chain)

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H L A D N A genotypes in Graves' disease 54 1

(Gustafsson el al., 1984); (iii) the Apal fragment of pII-a-5 (DQa chain) (Schenning et al., 1984). The DQB chain probe primarily detects DQP sequences, but with some cross- hybridization to non-DQP-chains. The DRJ chain probe is relatively specific for DRJ chain (Bidwell & Jarrold, 1986). The DQa probe is known to hybridize strongly to both DQa and DXa sequences (Spielman er al., 1984). Probes were labelled to a specific activity of lo9 cpm/pg by the oligonucleotide primer method (Feinberg & Vogelstein, 1983).

Prehybridization and hybridization were performed at 65°C in 6 x standard sodium citrate solution (SSC; 1 x SSC=O.15 M NaCI, 0.015 M Na citrate), 5 x Denhardt's solution, 0.5% sodium dodecyl sulphate (SDS) with 250 pg/ml denatured salmon sperm DNA. Ten percent dextran sulphate was used in the hybridization solution. After hybridization, filters were first rinsed in 2 x SSC, 0.1 YO SDS, twice, at room temperature, then washed in the same solution at 65°C for 30 min. Washing was then performed in 0.5 x SSC, 0.1 Yo SDS at 65°C for 60 min, followed by four 15 min washes in 0.1 x SSC, 0. I Yo SDS at 65°C. After air drying, autoradiography was performed for three days at

Typing for DR specificities 1-7 was performed on peripheral blood B lymphocytes using a standard microlymphocytotoxicity technique and well characterized sera (UK Transplant Service). DRw6 assignments were tentative in DR3/w6 subjects because of cross-reaction between DRw6 sera and DR3.

test when the smallest expected value in a 2 x 2 association table was five or greater, and by Fisher's exact test when this was not the case. Significance levels (P-values) were corrected by multiplying by the number of alleles at each locus (PJ. Relative risk (RR), and 95'Y0 confidence limits for relative risk, were calculated using thqmethod of Woolf, with the Haldane modification for small numbers (Thomson, 198 1).

Genotype frequency analysis was performed according to the 'antigen genotype frequencies among patients' (AGFAP) method of Thomson (Thomson, 1983). This is based on a simple two-locus association model. The first locus is the marker locus, and the second is the disease susceptibility locus with which the marker is in linkage disequili- brium; in the present study, the marker locus was defined using DRJchain RFLPs. Using this technique, it is possible to calculate the expected numbers of affected individuals in each HLA genotype class under various models of inheritance. These may then be compared with the observed number of subjects in each of these classes (see Appendix for details).

- 70°C.

Statistical analysis was by the

RESULTS

DRP gene probing

Seven DRP restriction fragment patterns were defined. The frequencies of these patterns in non-DR typed Graves' disease patients and control subjects are shown in Table 1. This table also shows the DR antigen with which each pattern is known to be associated (Kohonen-Corish & Sejeantson, 1986; Carlsson et al., 1987; Bidwell et al., 1988; Fletcher et al., 1988). A pattern of 10+7.0+4.0 kb DRP fragments was increased in frequency in patients us control subjects (66% us 32%; RR=3.9 (95Y0 confidence limits: 1.4-1 I ) ; P < 0.0 1; P, < 0.06). No other DRP RFLP differed significantly in frequency.

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542 J . Fletcher et al. Table I . DRB Taql RFLP frequencies (percentages) in Graves'

disease patients and control subjects

6+4.5 (DRI) 12+2+1.5 (DR2) 10+7+4 (DR3/w6) 12+7+4 (DR3/w6) l6+6+ 5.5+ 2 3 (DR4) 12+6.5+4 (DR5) 16+ 7+4+ 2.5 (DR7)

RR

0.6 0.8 3.9 1.3 1.3 1.1 0.8

P

NS NS c 0.0 I

NS NS NS NS

The DR antigen to which each pattern corresponds is indicated

RR. relative risk. P, significance level. tb. DNA fragment size in kilobases.

in brackets (see Discussion).

Table 2. DQP BamHl RFLP frequencies (percentages) in Graves' disease patients and control subjects

Fragments (kb)

6.2 + 3.2 7.0 + 3.0 7.0+4.0 I2 7,0+3.7 7.0 4.0 + 3.2

Patients (n = 33)

Controls (n=33)

9 (27) 14 (42)

13 (39) 12 (36)

2 (6) 6 (18)

5 (15)

RR

I .o 0.9 6.2 0.6 0.7 0.6 I .o

P

NS NS

c o a l I NS NS NS NS

RR, relative risk. P, significance level. kb, DNA fragment size in kilobases.

DQP gene probing

With the DQP probe and BamHI, seven distinct restriction fragment patterns were defined. These RFLPs are well recognized, and their associations with DR antigens have been established (Bohme er a/., 1985; Carlsson et a/., 1987; Fletcher er a/., 1988). Table 2 shows the frequencies of these patterns in non-DR typed Graves' disease patients and control subjects. The 7.0 +4.0 kb pattern was increased in frequency in patients us control subjects (55% us 15%; RR=6-2 (95% confidence limits: 2.1-19); P<0.001; Pc<0-006). No other DQB pattern differed significantly in frequency.

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HLA D N A genotypes in Graves’ disease

Table 3. DQa restriction fragment frequencies (per- centages) in Graves’ disease patients and control

subjects

543

Fragment Patients Controls (kb) (n=36) (n=25) RR P

2.6 10 (28) 8 (32) 0.8 NS 4.6 27 (75) 9 (36) 5.0 ~ 0 . 0 0 5 5.5 18 (50) 13 (52) 0.9 NS 6.2 13 (36) 14(56) 0.5 NS 6.8 0 (0) 2 (8) 0.1 NS

RR. relative risk. P, significance level.

kb, DNA fragment size in kilobases.

Table 4. DXa TaqI genotype and allele frequencies in Graves’ disease patients and control subjects

Genotypes Alleles

n 2.1/2.1 2.1/1.9 1.9/1.9 2.1 1.9

Patients 36 9 I8 9 0.5 0.5 Controls 25 5 8 I2 0.36 0.64

-~

1.9 and 2.1 refer to the size of the DXa Taql allelic

Genotype frequency difference: x2 (2 d.f.) = 3.55.

Allele frequency difference: x2 ( I d.f.) =2.34.

DNA fragments in kilobases.

P=NS.

P= NS.

DQa and DXa polymorphisms

Seven hybridizing fragments (1.9,2.1, 2.6,4.6, 5.5, 6.2 and 6-8 kb) were found with TaqI and the DQa probe. The 1.9 and 2.1 kb fragments are alleles of the DXa gene (Amar et d., 1987). The associations of the DQa fragments with DR antigens have been established (Carlsson et al., 1987; Bidwell et d., 1988; Fletcher et af., 1988). The frequencies of the DQa fragments in non-DR typed Graves’ patients and control subjects are shown in Table 3. The 4.6 kb fragment was increased in patients us control subjects (75% us 36%; RR = 5.0 (95% confidence limits: 1.7-1 5) ; P < 0-005; Pc < 0.02). No other fragment differed significantly in frequency.

DXa genotype and allelic frequencies in non-DR-typed Graves’ disease patients and control subjects are shown in Table 4. No significant differences in genotype or allelic frequencies were found.

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544 J . Fletcher et al.

D R3-positive subjects

In order to determine whether the two DR3-related DRJ RFLPs differed in their associations with Graves’ disease, DRJ RFLPs were compared in 21 DR3-positive Graves’ disease patients and 32 DR3-positive control subjects. The DRP 10+ 7.0 +4.0 kb fragment pattern showed close correlation with DR3, being present in 20/21 (95%) DR3- positive patients and in 29/32 (91%) DR3-positive control subjects ( P = N S ) . The D R j 12+ 7-0+4.0 kb fragment pattern was non-significantly reduced in frequency in DR3- positive patients compared with DR3-positive control subjects (1/21 ( 5 % ) us 7/32 (22%); P= NS). No other D R j RFLPs differed significantly in frequency between DR3-positive patients and control subjects.

DRP genofype analysis

Non-DR-typed patients were grouped into three genotype categories: DRJ3/3 homozy- gotes, DRJ3/X heterozygotes and DRPXIX homozygotes, where DRJ3 is the TaqI 10+7+4 kb RFLP, and DRJX is any other RFLP. The observed frequencies for each of these genotype categories were then compared with those expected under recessive expectations (Table 5 ) . This comparison rejects recessive inheritance of the DRP3- associated disease gene (P=O-Ol).

Table 5. ORB genotype frequencies in Graves’ patients

313 3/X X/X P

Observed 0 23 12 Expected 3.8 15.4 15.8 =0.01

Observed OR@ genotype frequencies and expected frequencies for simple recessive inheritance calculated by the AGFAP method.

3, DR3-associated DRJ RFLP (TaqI 10+7+4 kb fragments).

X, any other RFLP. P. significance level of difference between

expected and observed frequencies of 3/X heterozygotes (normal approximation to I

binomial distribution).

DISCUSSION

DRP chain gene probing produces restriction fragment patterns which correlate very closely with serological DR specificities, but with TaqI restriction enzyme two patterns correspond to DR3 (and DRw6): 12+7+4 kb and 10+7+4 kb (Kohonen-Corish & Serjeantson, 1986; Carlsson et al., 1987; Bidwell er al., 1988; Fletcher ef at., 1988). The latter pattern is known to correspond to the B8-DR3 haplotype, while the former is associated with B18-DR3 (Serjeantson ef al., 1986; Bidwell et al., 1988). We found the

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H L A D N A genotypes in Graves’ disease 545

10+7+4 kb pattern to be increased in frequency in Graves’ patients compared with control subjects. After correction for the number of DRP alleles, this was non-significant at the 5 % level, but in view of the strong prior hypothesis of association between the B8- DR3 haplotype and Graves’ disease, there can be little doubt that this association is genuine. Semana et al. (1987) have also shown this association. In the present study, this RFLP was present in the great majority of both DR3-positive Graves’ patients and DR3- positive control subjects.

No evidence for an association of the 12+7+4 kb RFLP with Graves’ disease was found. None of the other DR/3 patterns differed significantly in frequency, either in the non-DR typed, or in the DR3-positive patients and control subjects, suggesting that the 30-50% of Graves’ disease patients who do not express DR3 are unlikely to be accounted for by another DR antigen.

The DQP chain 7+4 kb pattern and the DQa 4.6 kb fragment were both significantly increased in frequency in Graves’ patients compared with control subjects. These RFLPs are known to be in close linkage disequilibrium with DR3 (Bohme et al., 1985; Carlsson el al., 1987; Bidwell et al., 1988; Fletcher et al., 1988). No other DQa or DQ/3 RFLPs showed significant associations with Graves’ disease. No significant differences were seen for DXa genotype or allele frequencies. The 2.1 kb allele is, however, known to be associated with DR3 in both control subjects and in Graves’ disease patients (Weetman et al., 1988), which probably accounts for the non-significant increase in frequency of this allele in patients seen in the present study.

In conclusion, the associations defined in this study could all be accounted for by the known association of the B8-DR3 haplotype with Graves’ disease. No non-DR3-related RFLP showed an association.

There is controversy concerning the mode of inheritance of the HLA-linked component of Graves’ disease susceptibility. Studies of HLA haplotype combinations in affected sibling-pairs have shown a high degree of DR-identity, which excludes simple dominant inheritance, but which is consistent with simple recessive inheritance (Stenszky ef al., 1985). An alternative approach to the investigation of inheritance patterns in HLA-linked disorders is the analysis of HLA genotype frequencies in affected subjects (Thomson, 1983). HLA genotyping using serological techniques requires family studies because of the existence of the blank ‘specificity’. RFLP analysis allows direct HLA genotyping, however, without the need to study relatives.

We have identified a DRfi RFLP (10 + 7 + 4 kb), which was related to B8-DR3, and which was positively associated with Graves’ disease; no homozygotes for this RFLP were observed among the 23 patients that possessed it. This high frequency of heterozygosity was incompatible with simple recessive inheritance. The lack of any excess of DR3 homozygotes (determined by serological typing) among Graves’ patients, compared with control subjects, has been noted by others (Farid et al., 1980; Stenszky et al., 1985), although the latter study did find an increased frequency of B8 homozygosity in Graves’ patients.

The sibling-pair studies suggest, therefore, that a contribution from both HLA haplotypes is required for disease susceptibility, but the genotype data indicate that usually only one of these haplotypes bears DR3. It is possible, therefore, that Graves’ susceptibility may be due to an interaction between two different HLA-linked genes (only one of which is in linkage disequilibrium with DR3). A possible precedent for HLA interaction exists in the case of insulin-dependent diabetes, where synergism between two

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546 J . Fletcher et al.

different HLA-linked genes (one associated with DR3 and the other with DR4) may account for the high frequency of DR3/4 heterozygotes in this condition (Rotter el al., 1983). If our interpretation is correct, it would appear that the non-DR3 susceptibility factor for Graves’ disease lies outside DQ-DR, because we did not find any association with a non-DR3-related DQa, DQP or DRP RFLP. The existence of an HLA-linked but non-DR-associated susceptibility gene might also account for the 30-50% of Graves’ patients who do not express DR3.

In conclusion, we have found associations of DR and DQ RFLPs with Graves’ disease which can be accounted for by linkage disequilibrium with B8-DR3, but did not identify any non-DR3-related RFLP associated with the condition. Analysis of HLA class I1 genotypes using DRP RFLPs indicates that simple models of inheritance for the HLA- linked component of Graves’ disease susceptibility may be inadequate.

APPENDIX

The expected frequencies of the three genotype classes under simple recessive inheritance are:

DRP genotypes

313 3/X x/x k2 2 k ( l - k ) (1-k)’

where k is the association parameter between marker and ‘disease’ allele. The maximum likelihood estimate for k for the recessive model is given by (2n2+nl)/

2 N , where n2, nl and no are the observed frequencies for the 313, 3/X and X/X classes, respectively, and N=nz+nl +no.

In this study, k = 23/70 = 0.329. The significance (two-tailed) of the excess of observed DRP3/X heterozygotes over

those expected under simple recessive inheritance was then determined using the binomial test (normal approximation).

ACKNOWLEDGEMENTS

This work was supported by an MRC Project Grant. JF is an MRC Training Fellow. The cDNA clones were kindly provided by Professor P.A. Peterson, Uppsala, Sweden.

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