HLA-DMB gene and HLA-DRA promoter region polymorphisms in Australian multiple sclerosis patients

HLA-DMB Gene and HLA-DRA Promoter Region Polymorphisms in Australian Multiple Sclerosis Patients Bruce H. Bennetts, Suzy M. Teutsch, Marc McW. Buhler, Robert N. S. Heard, and Graeme J. Stewart ABSTRACT: The MHC region has been shown to contain a susceptibility locus for multiple sclerosis (MS). While the strongest association to date has been between HLA-DRB1*1501 and MS, the exact nature of the MHC association in MS remains unclear. Two candidate polymorphic loci within the MHC class II region, the HLADMB gene and the HLA-DRA promoter, which lie close to HLA-DRB1, were therefore examined in an Australian MS population. The HLA-DMB*0103 phenotype was increased in the MS patients (46% vs. 30%) and the frequency of the HLA-DRA promoter A allele was also increased (81% vs. 68%). When the subjects were stratified into HLA-DRB1*1501 positive and negative individuals these associations were not significantly different. This is a result of the strong linkage disequilibrium between HLA-DRB1*1501 and both HLA-DMB*0103 and the HLA-DRA promoter A allele. The complete linkage between DRB1*1501 and the HLA-DRA pro-

INTRODUCTION Multiple sclerosis (MS) is a disease with a strong genetic component. There is unequivocal evidence for familial clustering [1] with a 20 – 40 fold increased risk of the disease amongst biological first-degree relatives of individuals with MS [2] and adoption studies indicate that familial aggregation of MS is largely genetically determined [3]. The MHC class II region contains the best described MS susceptibility loci, confirmed in association studies and genome screening [4 – 6], contributing an estimated 17– 60% of the genetic component of MS [7]. However, despite the first description of an MHC Class From the Department of Immunology, Westmead Hospital, Westmead 2145, Australia. Address reprint requests to: Graeme Stewart, Associate Professor, Department of Immunology, Westmead Hospital, Westmead 2145, Australia; Tel: 61 2 9845-6791; Fax: 61 2 9891-3889; E-Mail: stewartg@westmed. wh.usyd.edu.au. Received March 19, 1999; accepted April 21, 1999. Human Immunology 60, 886 – 893 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.

moter A allele indicates that the MS susceptibility haplotype (DRB1*1501-HLA-DQB1*0602-HLA-DQA1* 0102) can be extended out to promoter of the HLA-DRA locus. Interactions between both HLA-DMB and the HLA-DRA promoter and other reported MS susceptibility loci were examined (TCRBV polymorphisms, HLADQA1 and HLA-DQB1). Some interactions between specific TCRBV polymorphisms and the HLA-DRA promoter were observed, which is consistent with other published reports suggesting an epistatic interaction between TCRBV and HLA-DRB1. Human Immunology 60, 886 – 893 (1999). © American Society for Histocompatibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: HLA-DMB; HLA-DRA promoter; Multiple sclerosis; susceptibility; association

II/MS link more than 20 years ago [8, 9] the exact location of the MS gene(s) and its/their functional significance remains to be elucidated. Strong linkage disequilibrium (LD) between genes within the MHC region has hindered the identification of a single MS susceptibility locus; the association has been narrowed to the chromosomal segment encoding the haplotype HLADRB1*1501, -DQB1*0602 and -DQA1*0102 [10] with possible extension to the centromeric TAP2 locus [11]. In the past five years several new genes located within the class II region have been shown to have a vital role in antigen presentation (TAP1, TAP2, LMP2, LMP7, HLA-DMA, and HLA-DMB) and may have a functional role in the aetiology of MS. TAP1 and 2 and LMP2 and 7 have a key role in the processing and transport of antigenic peptides for presentation by MHC class I molecules. HLA-DMA and -DMB are essential components 0198-8859/99/$–see front matter PII S0198-8859(99)00054-3

HLA-DMB and -DRA Promoter Polymorphisms in MS

for the normal presentation of peptides by MHC class II molecules. Six HLA-DMB alleles (*0101-5 and *Val314) have been described [12–15]. An alternative hypothesis for the role of MHC genes in MS susceptibility is that the quantitative expression of MHC antigens is altered, rather than the specific structure of the putative MHC molecule. This may account for the exacerbation of the disease seen with the treatment of ␥-interferon (IFN) [16] and the potential beneficial effects of ␤-IFN [17]. The expression of class II antigens is regulated by their promoter regions and the factors that bind to these regions. Polymorphisms have been described within the promoter regions of a number of class II genes, including: HLA-DRA [18], -DRB [19], -DQB1 [20], and -DQA1 [21]. The HLA-DRA gene, unlike other class II genes has limited polymorphism in the structural portion of the gene [18] and thus little attention has been given to it as a potential candidate gene in MS susceptibility. Given the high likelihood of involvement of the MHC class II region in MS susceptibility and the potential functional role of these genes in disease aetiology, polymorphisms in the HLA-DMB structural gene and in the -DRA promoter region were investigated for associations in an Australian MS population. The results were also stratified with respect to T cell receptor polymorphism (TCRBV) in view of the likely epistatic interaction between susceptibility loci and the known biologic intimacy of MHC and T cell receptor. METHODS MS Patients and Control Subjects One hundred and one Australian MS patients with relapsing/remitting disease were selected by established criteria [22]. All patients had clinically definite or laboratory supported definite MS, with a disease duration of between 2 and 15 years, and a disability on the Kurtzke expanded disability status scale (EDSS) from 2 to 5 [23]. One hundred and two healthy volunteers were gathered from Westmead Hospital staff or partners of MS subjects. The ethnic origin of all patients and controls was European Caucasian, mainly Northern European (91% MS, 87% controls). An additional 8 known HLADRB1*1501 positive controls were genotyped to improve the comparison within the HLA-DRB1*1501 positive individuals. HLA Class II Typing HLA-DR serological typing, PCR-SSP typing and HLADRB1*15/16 genotyping was performed by the NSW Red Cross Tissue Typing Laboratory, Sydney. Additional genotyping was performed at Westmead of HLA-DQA1 and -DQB1 alleles using PCR-RFLP [24], and of

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TCRBV germline polymorphisms (Buhler et al., submitted for publication). DNA Extraction DNA was extracted from whole blood and from frozen white blood cells by a rapid salting out method [25]. PCR-RFLP Genotyping HLA-DMB. The third exon of the HLA-DMB gene was amplified using previously described primers [12]. A 20 ␮l reaction was prepared containing 0.1-1.0 ␮g of genomic DNA, 100 ng of HLA-DMB exon 3 specific primers, 200 ␮M dNTPs, 10 mM Tris (pH 8.3 at 25°C), 50 mM KCl, 4 mM MgCl2, and 1.5 U AmpliTaq (Perkin-Elmer, Norwalk, CT) mixed with an equal volume of TaqStart™ Antibody (Clontech, Palo Alto, CA) to prevent nonspecific amplification. Capillary PCR was performed using the following temperature profile: 1 cycle of 95°C for 3 min, 60°C for 15 s, 72°C for 15 s; 4 cycles of 94°C for 15 s, 60°C for 15 s, 72°C for 15 s; 35 cycles of 94°C for 3 s, 60°C for 3 s, 72°C for 15 s. The 285 bp PCR product was digested with ApaL I, Bsr I [15] and Hha I instead of Hinpl I, to allow identification of the HLA-DMB*0101-5 alleles. When HLA-DMB*0101 was present, samples were additionally amplified with HLA-DMB exon 2 primers [12] as described above. The 279 bp PCR product was digested with Sau3AI to identify the HLA-DMB*Val314 allele [14]. DMB*0101 was distinguished by 154 bp, 88 bp, 19 bp, 18 bp restriction fragments, and DMB*Val314 by 154 bp, 106 bp, 19 bp restriction fragments. Resolution of HLA-DMB*0101/0104 from HLADMB*0103/0105 was determined by PCR-SSCP analysis. Briefly, 3 ␮l of PCR products were mixed with an equal volume of a denaturing loading buffer (0.5% bromophenol blue, 0.5% xylene cyanol in 100% formamide), heated at 95°C for 5 min and quickly chilled on ice. Samples were loaded on to a 10% polyacrylamide gel (Hoefer, San Francisco, CA) and electrophoresed in 1 ⫻ TBE at 0°C for 2 h 45 min, or until both dye fronts had migrated off the gel. The gel was silver-stained to visualise SSCP band patterns. HLA-DRA promoter. HLA-DRA promoter fragments were initially amplified using primers described by Pinet et al. [18]. A 20 ␮l reaction was prepared containing 0.1–1.0 ␮g of genomic DNA, 100 ng of HLA-DRA promoter specific primers, 200 ␮M dNTPs, 10 mM Tris (pH 9 at 25°C), 50 mM KCl, 0.1% Triton-X 100, 2 mM MgCl2 and 1.5 U Taq polymerase (Promega, Madison, WI). PCR was performed in a capillary thermocycler (Corbett Research, Australia) using the following temperature profile: 1 cycle of 95°C for 3 min, 55°C for 15 s,

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72°C for 1 min; 39 cycles of 94°C for 15 s, 55°C for 15 s, 72°C for 1 min. The 775 bp PCR product was digested with the restriction enzymes Dde I and Pvu II (NEB, Beverley, MA) to determine the HLA-DRA promoter A and B alleles (Table 1). To improve the efficiency of the genotyping, additional primers were designed (forward 5⬘ CTG CCA AAA TTC AGA CAA 3⬘ and reverse 5⬘ TTG CTA GGG GAA GGG TTC TT 3⬘) to amplify a smaller fragment. Reactions of 20 ␮l were prepared, as described above, except that they contained 4 mM MgCl2. Capillary PCR was performed, using the following temperature profile: 1 cycle of 95°C for 3 min, 60°C for 15 s, 72°C for 15 s; 4 cycles of 94°C for 15 s, 60°C for 15 s, 72°C for 15 s; 35 cycles of 94°C for 3 s, 60°C for 3 s, 72°C for 15 s. The 345 bp PCR product was digested with Dde I and Pvu II to determine the HLA-DRA promoter A allele (undigested) and the B allele (Dde I: 185 bp, 160 bp, Pvu II 262 bp, 83 bp). Statistical Analysis Associations between HLA-DMB alleles, HLA-DRA promoter alleles and MS were examined separately using the chi-squared test. Relative risks were calculated using Woolf’s formula and Haldane’s modification when necessary [26] to determine if other genetic markers (HLADQA1, -DQB1, TCRBV) conferred additional risk. pValues were corrected by multiplying by the number of comparisons made (Bonferoni inequality method). Linkage disequilibrium was determined by calculating delta values [27]. Logistic regression analysis was performed using GLIM 4 (1992, Royal Statistical Society NAG, Banbury Road, Oxford) to determine if there were significant interactions between DRA promoter genotypes and HLA-DR, -DQA1, -DQB1 and TCRBV genetic markers. RESULTS HLA-DMB The HLA-DMB*0103 phenotype was significantly increased in MS patients (46% vs. 30%, pnc ⫽ 0.026) (Table 1). However, there were no significant differences or noticeable trends when the patients and the controls were stratified into HLA-DRB1*1501 positive and negative individuals (Table 1). This suggests that the increase in the HLA-DMB*0103 phenotype was secondary to the HLA-DRB1*1501 association, and was due to the strong linkage disequilibrium between HLADMB*0103 and HLA-DRB1*1501 (⌬ ⫻ 104 ⫽ 6.63, p ⫽ 4.82 ⫻ 10⫺6). However, there was no single HLADMB allele which was present in all HLA-DRB1*1501 positive individuals. Within the 11 individuals who were homozygous for HLA-DRB1*1501, 7 had the

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HLA-DMB*0101/*0103 genotype, 2 had the HLADMB*0101/*0101 genotype, 1 had the HLADMB*0103/*0103 genotype and 1 had the HLADMB*0103/*0104 genotype, suggesting that linkage disequilibrium was weak. HLA-DRA Promoter There was a significant increase in the frequency of both the HLA-DRA promoter A allele (pnc ⫽ 0.003) and the HLA-DRA promoter A/A genotype (pnc ⫽ 0.011) in the MS patients (Table 2). However, when MS and control subjects were stratified according to HLA-DRB1*1501 positive or negative phenotype there was little difference in the distribution of the HLA-DRA promoter A allele between either group (Table 2). There was significant linkage disequilibrium between HLA-DRB1*1501 and the HLA-DRA promoter A allele (⌬ ⫻ 104 ⫽ 615, p ⫽ 0.001). All of the 11 HLA-DRB1*1501 homozygotes had the HLA-DRA promoter A/A genotype and none of the HLA-DRB1*1501 positive individuals had the B/B genotype (Table 2) suggesting strong linkage disequilibrium. Epistatic Interaction Relative risks were used to identify potential interactions between the HLA-DRA promoter and other putative MS susceptibility loci (data not shown). These were then investigated using logistic regression analysis, which found that there were significant interactions between HLA-DRA promoter genotypes and TCRBV3S1, TCRBV10S1 and TCRBV15S1 (Table 3). DISCUSSION This study has examined two important candidate loci within the MHC, a potential role for which should be examined in several populations of MS patients before inclusion or exclusion as being relevant to the disease. The only published study of HLA-DMB polymorphisms in MS involved 63 Italian patients in whom no association was found [28]. Associations between DRA promoter polymorphisms and MS have not been previously reported. HLA-DMA and DMB, have been shown to be crucial for normal antigen processing and presentation of proteins by MHC class II molecules [29 –31]. Mutant cell lines with defects for MHC class II-mediated antigen presentation can be restored to normal phenotype upon transfection with HLA-DMA and -DMB genes [29]. Normally, close to 100% of MHC class II molecules are occupied with peptides derived from exogenous proteins. However, up to 50% of the total MHC class II molecules in these antigen processing mutants have instead a set of peptides derived from the Ii known as CLIP (class II

HLA-DMB and -DRA Promoter Polymorphisms in MS

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TABLE 1 HLA-DMB Frequencies A. All individuals Phenotype Frequency (%) DMB Alleles

MS (N ⫽ 101)

*0101 *0102 *0103 *0104 *0105 *Val314

Controls (N ⫽ 102)

93 8 46 7 0 4

94 12 30 6 0 2

p 0.760 0.358 0.026 0.760 0.400

Allele Frequency (%) DMB Alleles

MS (2N ⫽ 202)

Contols (2N ⫽ 204)

0.6683 0.0396 0.2376 0.0347 0 0.0198

0.7304 0.0588 0.1716 0.0294 0 0.0098

*0101 *0102 *0103 *0104 *0105 *Val314

p 0.173 0.371 0.099 0.764 0.404

B. HLA-DRB1*1501 positive individuals Phenotype Frequency (%) DMB Alleles

MS (N ⫽ 59)

*0101 *0102 *0103 *0104 *0105 *Val314

Controls (N ⫽ 25)

90 8 59 7 0 0

84 4 56 4 0 0

p 0.451 0.467 0.778 0.623

Phenotype Frequency (%) DMB Alleles

MS (2N ⫽ 118)

Controls (2N ⫽ 50)

0.6102 0.0424 0.3136 0.0339 0 0

0.6000 0.0200 0.3600 0.0200 0 0

*0101 *0102 *0103 *0104 *0105 *Val314

p 0.902 0.475 0.558 0.628

C. HLA-DRB1*1501 negative individuals Phenotype Frequency (%) DMB Alleles *0101 *0102 *0103 *0104 *0105 *Val314

MS (N ⫽ 42)

Controls (N ⫽ 77)

98 7 26 7 0 10

97 14 22 6 0 3

p 0.943 0.248 0.613 0.892 0.099

Phenotype Frequency (%) DMB Alleles *0101 *0102 *0103 *0104 *0105 *Val314

MS (2N ⫽ 84)

Controls (2N ⫽ 154)

0.7500 0.0357 0.1310 0.0357 0 0.0476

0.7727 0.0714 0.1104 0.0325 0 0.0130

p 0.693 0.263 0.638 0.894 0.103

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TABLE 2 DRA Promoter Results in MS and Control Patients Gene Frequency DRA promoter Allele All individuals MS n ⫽ 101 Controls n ⫽ 102 DRB1*1501 positive MS n ⫽ 62 Controls n ⫽ 33 DRB1*1501 negative MS n ⫽ 39 Controls n ⫽ 77

Genotype Frequency (%)

A

B

A/A

A/B

B/B

pnc

0.8069 0.6765 p ⫽ 0.003

0.1931 0.3235

65 45

31 45

4 10

0.011

0.8952 0.8485 p ⫽ 0.348

0.1048 0.1515

79 70

21 30

0 0

0.312

0.6667 0.6234 p ⫽ 0.517

0.3333 0.3766

44 38

46 49

10 13

0.801

associated invariant chain peptides). This suggests that CLIP is not removed from the class II molecules thus preventing the binding of antigenic peptides. It is thought that the DM molecule has a role as a catalyst for peptide exchange, selecting for the most stable class II:

peptide complexes [32], as HLA-DM has been shown to be largely absent from the plasma membrane and to have an obstructed cleft with in vitro studies showing there is little binding of synthetic peptides to DM [33]. While it is unclear as to whether the polymorphisms in the DM

TABLE 3 Significant interactions detected by logistic regression analysis between DRA promoter genotypes and TCRBV3S1*1, 10S1*1 and 15S1*1 alleles, expressed as odds ratios and showing 95% confidence intervals DRA promoter genotype

TCRBV3S1*1 negative positive

A/A

A/B

B/B

1

0.13 (95% CI ⫽ 0.03–0.54) 0.43 (95% CI ⫽ 0.16–1.2)

0.06 (95% CI ⫽ 0.01–0.54) 0.40 (95% CI ⫽ 0.07–2.4)

0.58 (95% CI ⫽ 0.23–1.5)

p ⫽ 0.029. DRA promoter genotype

TCRBV10S1*1 negative positive

A/A

A/B

B/B

1

0.64 (95% CI ⫽ 0.12–3.4) 0.52 (95% CI ⫽ 0.18–1.5)

—

1.04 (95% CI ⫽ 0.37–3.0)

0.07 (95% CI ⫽ 0.007–0.69)

p ⫽ 0.038. DRA promoter genotype

TCRBV15S1*1 negative positive p ⫽ 0.043.

A/A

A/B

B/B

1

0.38 (95% CI ⫽ 0.06–2.2) 0.43 (95% CI ⫽ 0.14–0.13)

—

0.96 (95% CI ⫽ 0.34–2.9)

0.06 (95% CI ⫽ 0.006–0.62)

HLA-DMB and -DRA Promoter Polymorphisms in MS

genes have a direct effect in altering peptide presentation of an MS antigen, their role in MS susceptibility is important to examine because of their location in the MHC class II region. The results for HLA-DMB support the Italian study [28], in that they did not find a major role for HLA-DM in MS susceptibility. However, there was a reduced frequency of the HLA-DMB*0103 allele in HLADRB1*1501 positive individuals in the Italian study (this may be due to a typographical error in their manuscript but it is still present after taking this into consideration) compared with the data in this report. The difference between the two MS association studies may lie in the origin of the individuals, given the predominantly Northern European origin of Australian MS patients. While linkage disequilibrium between HLADRB1*1501 and HLA-DMB*0103 was found, which has been previously shown by Djilali-Saiah et al. [14], this was not strongly consistent in the DRB1*1501 homozygotes suggesting considerable recombination between the two loci. Polymorphisms have been described in the promoter regions of the HLA-DRA, HLA-DQB1, HLA-DQA1, HLA-DRB and TNF genes, all of which are encoded in the MHC region on 6p21. Some of the established polymorphisms have mapped to areas known to be involved in the regulation of expression. Chloramphenicol acetyltransferase (CAT) assays have been used to demonstrate marked allelic differences in the promoter strengths in nine HLA-DRB promoter alleles [19] and two DQB1 promoter alleles [20]. While this study found an association between MS and the HLA-DRA promoter A allele, there was strong linkage disequilibrium this allele and HLA-DRB1*1501 making it difficult to establish it as the cause of the MHC association. However, this does not diminish the need for to investigate polymorphisms within the promoter/regulatory regions of the MHC class II genes as the cause of MHC susceptibility in autoimmune disease. While there were associations between MS and either HLA-DMB gene or HLA-DRA promoter polymorphisms in this study, these were secondary to the association with the HLA-DRB1*1501, HLA-DQB1*0602, HLA-DQA1*0102 haplotype. Linkage disequilibrium between HLA-DRB1*1501 and these two loci reinforces the problem of splitting the MS MHC susceptibility haplotype. The HLA-DMB gene lies centromeric to HLA-DRB1, near HLA-DP, while the HLA-DRA promoter lies telomeric to HLA-DRB1. This is the first demonstration of an association with an MHC class II promoter and MS. The data suggests that there may be complete linkage disequilibrium between HLADRB1*1501 and the A allele of the HLA-DRA pro-

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moter. This means that the HLA-DRB1*1501, HLADQB1*0602, HLA-DQA1*0102 haplotype should be extended in the telomeric direction to include the HLADRA promoter. However, the less than 100% linkage disequilibrium between any of the HLA-DMB alleles and HLA-DRB1*1501 suggests that this region may not be part of the primary MS susceptibility haplotype. This is consistent with there being a recombination hotspot between TAP1 and TAP2 [34], which lies between the HLA-DMB and HLA-DRB1 loci. There is evidence to suggest that MS susceptibility loci may work in an epistatic manner [35]. It is important to determine whether there are two or more gene loci acting epistatically within the MHC region and/or gene locus on other chromosomes which increase the relative risk of MS. Evidence suggesting an epistatic interaction between HLA-DRB1*1501 and T cell receptor genotype has been found in multiple case families [36, 37] and in some population association studies [38 – 40]. The identification of some modest interactions between the TCRBV3S1, TCRBV10S1, TCRBV15S1 loci and DRA promoter genotypes (Table 3) is encouraging and warrants further investigations of interactions between the MHC region and the TCRBV region. However, within the MHC region, the data showed no evidence for an epistatic effect between HLA-DMB and HLA-DRB1. It is now suspected that the majority, if not all potential MS susceptibility genes may confer a relatively low genotype relative risk (GRR) and that these genes may act in an epistatic manner [35]. This has important methodological implications for studies such as this one. To investigate these potential epistatic interactions between putative MS susceptibility loci such as the MHC and the TCRBV, numerous comparisons have to be made. This necessitates the need for correcting for multiple comparisons before being able to reliably infer statistical significance. However, this conservative but appropriate approach may lead to type II statistical errors, particularly when given low GRRs. This may lead to the premature dismissal of putative MS susceptibility genes. The problems outlined above can be addressed in several ways. These include the publishing of full data of allele frequencies found by studies with different MS populations. This will allow MS researchers to observe trends in data from other studies which may be similar to their own, and then to test these in a new population with an a priori hypothesis or, where appropriate, to use meta analyses to assess a larger body of data. The role of the MHC region remains to be elucidated but its importance in MS susceptibility and the opportunity for therapeutic intervention remains unaltered.

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ACKNOWLEDGMENTS

This work has been supported by the National Multiple Sclerosis Society of Australia. HLA-DR typing was performed by the NSW Red Cross Tissue typing Laboratory. We would like thank Professor James McLeod, University of Sydney and Claudia Charlton for the recruitment and collection of blood from MS patients. The authors also wish to thank Dr. Karen Byth for help with the logistic regression analysis.

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