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HLA-DRB1 allele heterogeneity influences multiple sclerosis severity as well as risk in Western Australia
Journal of Neuroimmunology 219 (2010) 109–113
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
HLA-DRB1 allele heterogeneity influences multiple sclerosis severity as well as risk in Western Australia Jing-Shan Wu a, Ian James c, Wei Qiu a,e, Alison Castley b, Frank T. Christiansen b,d, William M. Carroll a, Frank L. Mastaglia a, Allan G. Kermode a,⁎ a Centre for Neuromuscular and Neurological Disorders, University of Western Australia; Department of Neurology, Sir Charles Gairdner Hospital, Queen Elizabeth II Medical Centre, Perth, Western Australia, Australia b Department of Clinical Immunology and Immunogenetics, PathWest Royal Perth Hospital, Perth, Western Australia, Australia c Centre for Clinical Immunology and Biomedical Statistics, Murdoch University and Royal Perth Hospital, Perth, Western Australia, Australia d School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Western Australia, Australia e Department of Neurology, the Third Affiliated Hospital of Sun Yet-sen University, Guangzhou, China
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Article history: Received 25 September 2009 Received in revised form 17 November 2009 Accepted 20 November 2009 Keywords: Multiple sclerosis Human Leukocyte Antigen (HLA) Susceptibility Multiple Sclerosis Severity Score
a b s t r a c t Susceptibility to multiple sclerosis (MS) has been consistently associated with the Human Leukocyte Antigen (HLA)-DRB1⁎1501 genotype, however effects on disease severity and clinical outcome have varied in different populations. We present the results of a high-resolution HLA-DRB1 genotyping and genotype–phenotype correlation study in a large West Australian MS cohort. Our findings indicate that in this population, which is of largely Anglo-Celtic and Northern European origin, HLA-DRB1⁎1501 is not only a strong determinant of disease risk but may also be associated with disease severity as measured by the Multiple Sclerosis Severity Score (MSSS), with the MSSS increasing by an estimated 0.51 per DRB1⁎1501 allele. We also found evidence that the HLA-DRB1⁎1201 allele is associated with less severe disease. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a heterogeneous demyelinating disease whose pathogenesis is thought to involve a complex interaction between genetic factors, which are likely to vary in different populations and ethnic groups, and environmental influences whose importance varies in different geographic locations. Genetic susceptibility is strongly associated with allelic variations at the Human Leukocyte Antigen (HLA)-DRB1 gene locus (Hafler et al., 2007; Lincoln et al., 2005). Among the over 600 HLA-DRB1 alleles sequenced to date, the dominant association is with the HLA-DRB1⁎1501 allele which is found in over 50% of MS cases in all Caucasian populations so far studied (Schmidt et al., 2007) and which is more likely to be transmitted through the maternal line (Ramagopalan et al., 2008b). Studies in Europe and North America have shown that HLA-DRB1⁎1501 increases the risk of developing MS in a dose-dependent manner with homozygotes having several times higher risk than heterozygotes (Barcellos et al., 2003). As well as its effect on MS susceptibility, DRB1⁎1501 has also been found to be associated with a younger age of onset (Hensiek et al., 2002), a greater
⁎ Corresponding author. Australian Neuromuscular Research Institute, Sir Charles Gairdner Hospital; Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Queen Elizabeth II Medical Centre, 6009, Perth, WA, Australia. Tel.: +61 8 93881865; fax: +61 8 93882149. E-mail address: [email protected] (A.G. Kermode). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.11.015
number of brain MRI lesions (Hauser et al., 2000), and a worse outcome (Barcellos et al., 2003; Okuda et al., 2009) in some populations. However the findings with regard to clinical course (Hillert et al., 1992; Olerup et al., 1989) and disease outcome have been inconsistent (Barcellos et al., 2006; Ramagopalan et al., 2008a; Runmarker et al., 1994). Disease susceptibility in HLA-DRB1⁎1501 carriers has recently also been shown to be influenced by synergistic interactions with the alternate parental (in trans) allele at the DRB1 locus (Dyment et al., 2005). Thus, in a study of a large MS cohort DRB1⁎1501/⁎0801 heterozygosity was found to confer a higher risk, comparable to that of DRB1⁎1501 homozygotes, while carriers of the DRB1⁎15/14 genotype had a lower disease risk (Barcellos et al., 2006). Such epistatic interactions may affect the disease phenotype and outcome as well as susceptibility. For example, in a pooled Canadian–Sardinian MS cohort the DRB1⁎01 allele was shown to confer a milder disease outcome when carried in combination with DRB1⁎15 (DeLuca et al., 2007). It has been proposed that the genetic influences on disease susceptibility and outcome may be intrinsically linked (Ramagopalan et al., 2008a). Additional studies in other populations may be helpful to investigate this association further. In the present study high-resolution HLA-DRB1 genotyping was performed in a cohort of 495 well-characterized West Australian MS patients from the Perth Demyelinating Diseases Database (PDDD) (Wu et al., 2008), and the influence of allelic heterogeneity on disease severity was analysed. To our knowledge, this is the first such
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comprehensive study on a large MS cohort from the Southern hemisphere. 2. Methods
and at least 5 cases for MSSS analyses. Among the considered alleles only HLA-DRB1⁎1501, DRB1⁎0301 and DRB1⁎0701 had more than 2 homozygous cases (39, 14 and 5, respectively) and analyses of dose effects and homozygosity were restricted to these alleles. The cohort was shown to be in Hardy–Weinberg equilibrium.
2.1. Research participants 3. Results A total of 504 consecutive patients from the PDDD who were enrolled during the period from November 2006 to December 2008 were reviewed. The original country of birth of the patient and the patient's parents were recorded. Nine patients were non-Caucasian and were excluded from the study. Among the remaining 495 subjects, 466 were diagnosed as clinically definite or probable MS according to the Poser criteria (Poser et al., 1983) or MS using the modified McDonald criteria (Compston et al., 2001); 425 were classified as relapsing–remitting MS (RRMS) (including secondaryprogressive) and 41 as primary-progressive MS (PPMS). The control group comprised 189 Caucasian individuals from the Busselton Community Health Study (Welborn, 1998). The protocol was approved by the Sir Charles Gairdner Hospital Human Research Ethics Committee, and informed consent was obtained from all participants.
3.1. Demographic and clinical data The female-to-male ratio in the study cohort was 3.2:1. The average age of onset was 35.3 years and the mean disease duration was 13.2 years (0–58 years). EDSS and MSSS scores as recorded at the time of the last clinical assessment were obtained in 434 (93.1%) and 431 (92.5%) patients, respectively. The mean EDSS score was 3.5 ± 2.4 and the mean MSSS was 4.1 ± 2.8. The MSSS was strongly associated with age-at-onset (p = 0.0004) and gender (p = 0.002), and was higher in males than in females (4.8 ± 2.7 vs 3.8 ± 2.8, p = 0.001). The association with age-at-onset remains strong (p = 0.0004) even after adjustment for gender and the potential confounding (but nonsignificant) effect of disease duration, with an estimated increase of 0.05/year of age.
2.2. HLA-DRB1 typing 3.2. HLA-DRB1 alleles and MS severity High-resolution 4-digit HLA-DRB1 genotyping was performed in the Department of Clinical Immunology and Immunogenetics PathWest, Royal Perth Hospital by DNA sequencing. The HLA-DRB1 typing was performed using a previously reported method (Sayer et al., 2001) with the following modifications. The DNA was extracted from whole blood using the QIAGEN BioRobot M48 machine using Magattract DNA Blood Mini M48 kit (192). The PCR was performed in a 30 μl volume and purified using Agencourt® AMPure® kits PCR purification system. Automated sequencing was carried out using ABI Big Dye Terminator chemistry on ABI Prism 3730 and 3730xl Genetic Analysers and HLA-DRB1 analysis was carried out using ASSIGNV4.0.1.36 (Conexio Genomics). 2.3. Clinical and laboratory data Patients were assessed clinically by two neurologists in the MS clinic (AGK and WMC) at the time of collecting blood samples. Data recorded included: gender, family history of MS, age-at-clinical onset, disease duration, clinical course, Extended Disability Status Scale (EDSS) score at last visit, Global MS Severity Score (MSSS) (Roxburgh et al., 2005), visual evoked potential (VEP) and MRI findings on the most recent studies and results of cerebrospinal fluid studies. The MSSS scores were calculated based on individual's EDSS adjusted for disease duration using the Global MSSS table (Roxburgh et al., 2005). 2.4. Statistical analysis Comparisons of carriage frequencies of alleles between cases and controls were carried out by case–control logistic regressions for overall multivariable analyses and Fisher exact tests for individual alleles. Comparisons of EDSS and MSSS scores across groups and in multivariable analyses were carried out via linear regression. Analyses were carried out with and without adjustment for gender with consistent results. Overall analyses for each outcome based on the multivariable models are presented along with the univariate analyses for each allele separately. p-values b 0.05 were considered statistically significant. Although the individual tests are not independent, those results withstanding crude Bonferroni adjustment are indicated where appropriate. A total of 34 different HLA-DRB1 alleles were detected in this study cohort. To avoid potential artifacts due to small samples we considered only those alleles carried by at least 5 individuals in the combined samples for case–control comparisons
The carriage frequencies of 25 HLA-DRB1 alleles in cases and controls and the associations of the 21 alleles satisfying our inclusion criteria with MSSS are shown in Table 1. The HLA-DRB1⁎1501 allele was strongly associated with disease risk in both univariate and multivariable analyses, and carriage of this allele was also marginally associated with a higher mean MSSS compared with non-carriers (4.3 vs 3.8, p = 0.04) (Table 1), though this association does not withstand multiple comparison correction. Gender stratification showed no
Table 1 Associations of HLA-DRB1 alleles with MSSS and carriage frequencies in the MS and control groups of subjects. HLADRB1
Case carriage Control carriage Odds p-valuea MSSS mean p-valueb frequency% (n) frequency% (n) ratio difference
⁎0101 ⁎0102 ⁎0103 ⁎0301 ⁎0401 ⁎0402 ⁎0403 ⁎0404 ⁎0405 ⁎0407 ⁎0701 ⁎0801 ⁎0901 ⁎1001 ⁎1101 ⁎1103 ⁎1104 ⁎1201 ⁎1301 ⁎1302 ⁎1303 ⁎1401 ⁎1501 ⁎1502 ⁎1601
11.8% (55) 1.5% (7) 4.7% (22) 23.6% (110) 11.2% (52) 1.3% (6) 1.5% (7) 8.4% (39) 1.3% (6) 0.4% (2) 18.0% (84) 5.8% (27) 1.1% (5) 1.5% (7) 6.0% (28) 0.9% (4) 4.3% (20) 2.6% (12) 7.5% (35) 5.4% (25) 3.9% (18) 2.6% (12) 54.5% (254) 0.1% (2) 3.2% (15)
18.0% (34) 2.7% (5) 2.1% (4) 22.8% (43) 19.1% (36) 2.1% (4) 1.1% (2) 8.5% (16) 0.0% (0) 3.2% (6) 29.6% (56) 3.7% (7) 4.8% (9) 1.1% (2) 12.7% (24) 3.7% (7) 1.1% (2) 4.8% (9) 10.6% (20) 10.1% (19) 1.1% (2) 3.2% (6) 19.6% (37) 2.1% (4) 3.2% (6)
0.61 0.56 2.29 1.05 0.53 0.60 1.43 0.99 inf 0.13 0.52 1.60 0.22 1.43 0.44 0.23 4.19 0.53 0.69 0.51 3.76 0.81 4.92 0.20 1.01
0.04 ns ns ns 0.01 ns ns ns ns 0.01 0.0015c ns 0.01 ns 0.01 0.02 0.05 0.15 ns 0.04 0.08 ns 10− 14c ns ns
− 0.23 − 0.40 − 0.75 − 0.20 − 0.76 0.21 0.03 − 0.19 − − 0.20 0.77 1.77 − 1.25 0.82 − 0.74 − 2.34 − 0.65 − 0.78 0.40 − 0.70 0.56 − 0.29
ns ns ns ns 0.1 ns ns ns − − ns 0.2 ns ns ns − ns 0.003 ns 0.15 ns ns 0.04 − ns
inf: unable to estimate OR. Alleles significantly associated with MSSS were showed in bold. a When HLA-DRB1 allele carriage frequencies were compared between MS cases and healthy controls. b When HLA-DRB1 allele associated mean MSSS were compared between carriers and non-carriers. c p-values remain significant after Bonferroni correction.
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significant difference in the HLA-DRB1⁎1501 MSSS effect for female or male carriers of DRB1⁎1501 (p = 0.6). Of the other alleles analysed the only correlation with MSSS was with HLA-DRB1⁎1201 which was associated with a lower mean MSSS (− 2.3, p = 0.003). This HLADRB1⁎1201 effect remained strong (p = 0.005) even in the multivariable analysis adjusting for the large number of non-significant other alleles, and is close to significant after the crude Bonferroni correction (p = 0.06). 3.3. Dose effect of HLA-DRB1⁎1501 We analysed the HLA-DRB1⁎1501 dose effect for association with disease risk and severity. While numbers were insufficient to distinguish statistically between any effect being dose-dependent rather than simply carriage of DRB1⁎1501, we noted an approximately linear trend in dose effect for both risk (as measured by the OR) and severity (Fig. 1A and B). The MSSS in HLA-DRB1⁎1501 carriers increased by an estimated 0.51 per allele (p = 0.016 uncorrected). 3.4. Other HLA-DRB1 allele combinations Of the other 15 allele combinations analysed, HLA-DRB1⁎1501/0801 had the highest estimated disease risk (OR 8.7, though with small samples this just fails to reach significance, p = 0.07), and was also associated with the highest estimated mean MSSS (5.3 ± 2.5, n = 10), but was not significant when compared with other genotypes
Fig. 1. Dose effect of HLA-DRB1⁎1501 on MS susceptibility and MSSS. A. Carriage of two copies of HLA-DRB1⁎1501 allele conferred a higher estimated disease risk (odds ratio OR = 9.3 relative to no copies) than one copy (OR = 4.5) when compared with healthy controls. B. The MSSS in HLA-DRB1⁎1501 carriers increased by an estimated 0.51 per allele (p = 0.016), with a possible linear dose effect.
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(p = 0.08) (Fig. 2). Although the association between MSSS and other genotypes was not significant, Fig. 2 shows the gradient for the different HLA-DRB1⁎1501 genotypes compared with nonHLA-DRB1⁎1501genotypes. 4. Discussion While the strong genetic association between the HLA-DRB1 genotype and MS susceptibility has been demonstrated consistently across populations, the association with disease severity and outcome has remained more controversial (Barcellos et al., 2003; Barcellos et al., 2006; Ramagopalan et al., 2008a) as environmental and lifestyle factors may also have important effects on the natural history of the disease. The State of Western Australia is ideally suited for genotype– phenotype studies of this type as it has a population of ∼ 2.2 million which is largely of Anglo-Celtic and Northern European origin and is relatively stable. Studies in such geographically isolated and genetically relatively uniform populations may have certain advantages in detecting genetic contributions to disease phenotype and outcome (Hammond et al., 1988) when compared with larger multi-national studies of more heterogeneous patient cohorts. In the present study cohort, nine out of the 504 MS cases (1.8%) were non-Caucasian and were excluded from the study. This figure is comparable to the figure of 3% for outliers in a recently published eigenvector analysis of a European–Australian population sample (McEvoy et al., 2009). In the present study we performed high-resolution (4-point) HLA-DRB1 genotyping to investigate the effects of individual alleles and allele combinations at the HLA-DRB1 locus on disease severity. High-resolution typing allows more reliable definition of allelic variants with different functions which may remain ambiguous with 2-point typing (e.g. the sub-alleles of HLA-DR04 and -DR11) (Listgarten et al., 2008; Svejgaard, 2008). In this population we found that HLA-DRB1⁎1501 is not only the dominant DRB1 allele associated with disease risk as reported in previous Australian studies (Stankovich et al., 2009; Stewart et al., 1997), but may also make an apparent dose-dependent contribution to disease severity, albeit weaker than its contribution to risk. The only other allele which was found to be associated with disease severity was the minor allele HLA-DRB1⁎1201. It is of interest that in previous studies using 2-point genotyping HLA-DRB1⁎12 was found to be associated with lower risk (Ballerini et al., 2004; Masterman et al., 2000),
Fig. 2. Association between HLA-DRB1⁎1501 genotype and MSSS. The MSSS was significantly higher in HLA-DRB1⁎1501/1501 carriers when compared with nonDRB1⁎1501 genotypes (XX/XX) (mean MSSS 4.8 vs 3.8, p= 0.03); HLA-DRB1⁎1501/0801 carriers had the highest mean MSSS of 5.3 but this was not significant when compared with HLA-DRB1⁎XX/XX (p = 0.08).
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whereas in another Italian study HLA-DR1⁎12 was associated with more severe MRI changes (Zivadinov et al., 2003). In the present study HLA-DRB1⁎1201 was found to be associated with a lower MSSS. Our findings are consistent with the notion that epistatic interactions between HLA-DRB1⁎1501 and the other in trans alleles at the DRB1 locus may have a modifying effect on disease severity. Our estimated severity gradient for the various HLA-DRB1⁎1501 genotypes is comparable to that previously found for disease risk in European-derived populations in the Northern Hemisphere (Barcellos et al., 2006; Ramagopalan and Ebers, 2009). These findings support the concept that allelic interactions at the HLA-DRB1 locus may have modifying effects both on MS risk and severity. There is also evidence for similar disease-modifying effects of HLA-DRB1 allele interactions in other autoimmune diseases such as type 1 diabetes mellitus (Price et al., 1999; Steenkiste et al., 2007) and sporadic inclusion body myositis (Mastaglia et al., 2009). The biological mechanisms which underlie these effects remain speculative. It is well known that the proteins encoded by the HLA-DR genes bind antigenic peptides for presentation to CD4+ T lymphocytes (Jones et al., 2006; Watts, 2004). Polymorphic variants of HLA-DRB1 may therefore have differential binding properties for disease-related peptides based upon their amino acid sequences and may thereby modify the risk of inducing the disease as well as its severity and clinical course. As the two alleles at the DRB1 locus are co-dominant and are both expressed, the proteins encoded may present different antigenic epitopes to the immune system. Alternatively, one allele may present a disease-associated epitope and the other could have other effects on the immune response by acting through cytokine networks. However, determining the influence of genetic factors on disease severity and outcome is likely to be more complicated as gene–environment interactions may also play a part. For example, vitamin D can regulate the expression of HLA-DRB1⁎1501 (Ramagopalan et al., 2009), and such epigenetic interactions could potentially affect disease expression. This could also explain our observation of a strong association of HLA-DRB1⁎1501 with disease risk but much weaker association with disease severity. In summary, the present findings indicate that in this MS population allelic heterogeneity at the HLA-DRB1 locus may have modifying effects on disease severity as well as susceptibility. Similar studies in other populations would be helpful in determining the contribution of genetic factors to disease severity and outcome. Funding The National Health and Medical Research Council of Australia and Multiple Sclerosis Research Australia provided a Training Scholarship to Dr Jing-Shan Wu. Dr. Wei Qiu was supported by an Australian Endeavour International Postgraduate Research Scholarship (EIPRS) and Postgraduate Award from The University of Western Australia. Acknowledgements Steve Pummer and the staff at the Department of Clinical Immunology and Immunogenetics, PathWest, Royal Perth Hospital kindly assisted with sample collection and the HLA-DRB1 genotyping. References Ballerini, C., Guerini, F.R., Rombola, G., Rosati, E., Massacesi, L., Ferrante, P., Caputo, D., Talamanca, L.F., Naldi, P., Liguori, M., Alizadeh, M., Momigliano-Richiardi, P., D'Alfonso, S., 2004. HLA-multiple sclerosis association in continental Italy and correlation with disease prevalence in Europe. J. Neuroimmunol. 150, 178–185. Barcellos, L.F., Oksenberg, J.R., Begovich, A.B., Martin, E.R., Schmidt, S., Vittinghoff, E., Goodin, D.S., Pelletier, D., Lincoln, R.R., Bucher, P., Swerdlin, A., Pericak-Vance, M.A., Haines, J.L., Hauser, S.L., 2003. HLA-DR2 dose effect on susceptibility to multiple sclerosis and influence on disease course. Am. J. Hum. Genet. 72, 710–716.
Barcellos, L.F., Sawcer, S., Ramsay, P.P., Baranzini, S.E., Thomson, G., Briggs, F., Cree, B.C., Begovich, A.B., Villoslada, P., Montalban, X., Uccelli, A., Savettieri, G., Lincoln, R.R., DeLoa, C., Haines, J.L., Pericak-Vance, M.A., Compston, A., Hauser, S.L., Oksenberg, J.R., 2006. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum. Mol. Genet. 15, 2813–2824. Compston, A., Edan, G., Goodkin, D., Hartung, H.P., Lublin, F.D., McFarland, H.F., Paty, D.W., Polman, C.H., Reingold, S.C., Sandberg-Wollheim, M., Sibley, W., Thompson, A., van den Noort, S., Weinshenker, B.Y., Wolinsky, J.S., McDonald, W.I., 2001. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann. Neurol. 50, 121–127. DeLuca, G.C., Ramagopalan, S.V., Herrera, B.M., Dyment, D.A., Lincoln, M.R., Montpetit, A., Pugliatti, M., Barnardo, M.C., Risch, N.J., Sadovnick, A.D., Chao, M., Sotgiu, S., Hudson, T.J., Ebers, G.C., 2007. An extremes of outcome strategy provides evidence that multiple sclerosis severity is determined by alleles at the HLA-DRB1 locus. Proc. Natl. Acad. Sci. U. S. A. 104, 20896–20901. Dyment, D.A., Herrera, B.M., Cader, M.Z., Willer, C.J., Lincoln, M.R., Sadovnick, A.D., Risch, N., Ebers, G.C., 2005. Complex interactions among MHC haplotypes in multiple sclerosis: susceptibility and resistance. Hum. Mol. Genet. 14, 2019–2026. Hafler, D.A., Compston, A., Sawcer, S., Lander, E.S., Daly, M.J., De Jager, P.L., de Bakker, P.I., Gabriel, S.B., Mirel, D.B., Ivinson, A.J., Pericak-Vance, M.A., Gregory, S.G., Rioux, J.D., McCauley, J.L., Haines, J.L., Barcellos, L.F., Cree, B., Oksenberg, J.R., Hauser, S.L., 2007. Risk alleles for multiple sclerosis identified by a genomewide study. N. Engl. J. Med. 357, 851–862. Hammond, S.R., McLeod, J.G., Millingen, K.S., Stewart-Wynne, E.G., English, D., Holland, J.T., McCall, M.G., 1988. The epidemiology of multiple sclerosis in three Australian cities: Perth, Newcastle and Hobart. Brain 111 (Pt 1), 1–25. Hauser, S.L., Oksenberg, J.R., Lincoln, R., Garovoy, J., Beck, R.W., Cole, S.R., Moke, P.S., Kip, K.E., Gal, R.L., Long, D.T., 2000. Interaction between HLA-DR2 and abnormal brain MRI in optic neuritis and early MS. Optic Neuritis Study Group. Neurology 54, 1859–1861. Hensiek, A.E., Sawcer, S.J., Feakes, R., Deans, J., Mander, A., Akesson, E., Roxburgh, R., Coraddu, F., Smith, S., Compston, D.A., 2002. HLA-DR 15 is associated with female sex and younger age at diagnosis in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 72, 184–187. Hillert, J., Gronning, M., Nyland, H., Link, H., Olerup, O., 1992. An immunogenetic heterogeneity in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 55, 887–890. Jones, E.Y., Fugger, L., Strominger, J.L., Siebold, C., 2006. MHC class II proteins and disease: a structural perspective. Nat. Rev. Immunol. 6, 271–282. Lincoln, M.R., Montpetit, A., Cader, M.Z., Saarela, J., Dyment, D.A., Tiislar, M., Ferretti, V., Tienari, P.J., Sadovnick, A.D., Peltonen, L., Ebers, G.C., Hudson, T.J., 2005. A predominant role for the HLA class II region in the association of the MHC region with multiple sclerosis. Nat. Genet. 37, 1108–1112. Listgarten, J., Brumme, Z., Kadie, C., Xiaojiang, G., Walker, B., Carrington, M., Goulder, P., Heckerman, D., 2008. Statistical resolution of ambiguous HLA typing data. PLoS Comput. Biol. 4, e1000016. Mastaglia, F.L., Needham, M., Scott, A., James, I., Zilko, P., Day, T., Kiers, L., Corbett, A., Witt, C.S., Allcock, R., Laing, N., Garlepp, M., Christensen, F.T., 2009. Sporadic inclusion body myositis: HLA-DRB1 allele interactions influence disease risk and clinical phenotype. Neuromuscul. Disord. Neuromuscular Disorders 19, 763–765. Masterman, T., Ligers, A., Olsson, T., Andersson, M., Olerup, O., Hillert, J., 2000. HLA-DR15 is associated with lower age at onset in multiple sclerosis. Ann. Neurol. 48, 211–219. McEvoy, B.P., Montgomery, G.W., McRae, A.F., Ripatti, S., Perola, M., Spector, T.D., Cherkas, L., Ahmadi, K.R., Boomsma, D., Willemsen, G., Hottenga, J.J., Pedersen, N.L., Magnusson, P.K., Kyvik, K.O., Christensen, K., Kaprio, J., Heikkila, K., Palotie, A., Widen, E., Muilu, J., Syvanen, A.C., Liljedahl, U., Hardiman, O., Cronin, S., Peltonen, L., Martin, N.G., Visscher, P.M., 2009. Geographical structure and differential natural selection among North European populations. Genome Res. 19, 804–814. Okuda, D.T., Srinivasan, R., Oksenberg, J.R., Goodin, D.S., Baranzini, S.E., Beheshtian, A., Waubant, E., Zamvil, S.S., Leppert, D., Qualley, P., Lincoln, R., Gomez, R., Caillier, S., George, M., Wang, J., Nelson, S.J., Cree, B.A., Hauser, S.L., Pelletier, D., 2009. GenotypePhenotype correlations in multiple sclerosis: HLA genes influence disease severity inferred by 1HMR spectroscopy and MRI measures. Brain 132, 250–259. Olerup, O., Hillert, J., Fredrikson, S., Olsson, T., Kam-Hansen, S., Moller, E., Carlsson, B., Wallin, J., 1989. Primarily chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities. Proc. Natl. Acad. Sci. U. S. A. 86, 7113–7117. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H., Tourtellotte, W.W., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227–231. Price, P., Witt, C., Allcock, R., Sayer, D., Garlepp, M., Kok, C.C., French, M., Mallal, S., Christiansen, F., 1999. The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol. Rev. 167, 257–274. Ramagopalan, S.V., Deluca, G.C., Degenhardt, A., Ebers, G.C., 2008a. The genetics of clinical outcome in multiple sclerosis. J. Neuroimmunol. 201–202, 183–199. Ramagopalan, S.V., Herrera, B.M., Bell, J.T., Dyment, D.A., Deluca, G.C., Lincoln, M.R., Orton, S.M., Chao, M.J., Sadovnick, A.D., Ebers, G.C., 2008b. Parental transmission of HLA-DRB1⁎15 in multiple sclerosis. Hum. Genet. 122, 661–663. Ramagopalan, S.V., Ebers, G.C., 2009. Epistasis: multiple sclerosis and the major histocompatibility complex. Neurology 72, 566–567. Ramagopalan, S.V., Maugeri, N.J., Handunnetthi, L., Lincoln, M.R., Orton, S.M., Dyment, D.A., Deluca, G.C., Herrera, B.M., Chao, M.J., Sadovnick, A.D., Ebers, G.C., Knight, J.C., 2009. Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1⁎1501 is regulated by vitamin D. PLoS Genet. 5, e1000369. Roxburgh, R.H., Seaman, S.R., Masterman, T., Hensiek, A.E., Sawcer, S.J., Vukusic, S., Achiti, I., Confavreux, C., Coustans, M., le Page, E., Edan, G., McDonnell, G.V., Hawkins, S., Trojano, M., Liguori, M., Cocco, E., Marrosu, M.G., Tesser, F., Leone, M.A.,
J.-S. Wu et al. / Journal of Neuroimmunology 219 (2010) 109–113 Weber, A., Zipp, F., Miterski, B., Epplen, J.T., Oturai, A., Sorensen, P.S., Celius, E.G., Lara, N.T., Montalban, X., Villoslada, P., Silva, A.M., Marta, M., Leite, I., Dubois, B., Rubio, J., Butzkueven, H., Kilpatrick, T., Mycko, M.P., Selmaj, K.W., Rio, M.E., Sa, M., Salemi, G., Savettieri, G., Hillert, J., Compston, D.A., 2005. Multiple Sclerosis Severity Score: using disability and disease duration to rate disease severity. Neurology 64, 1144–1151. Runmarker, B., Martinsson, T., Wahlstrom, J., Andersen, O., 1994. HLA and prognosis in multiple sclerosis. J. Neurol. 241, 385–390. Sayer, D., Whidborne, R., Brestovac, B., Trimboli, F., Witt, C., Christiansen, F., 2001. HLADRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors. Tissue Antigens 57, 46–54. Schmidt, H., Williamson, D., Ashley-Koch, A., 2007. HLA-DR15 haplotype and multiple sclerosis: a HuGE review. Am. J. Epidemiol. 165, 1097–1109. Stankovich, J., Butzkueven, H., Marriott, M., Chapman, C., Tubridy, N., Tait, B.D., Varney, M.D., Taylor, B.V., Foote, S.J., Kilpatrick, T.J., Rubio, J.P., 2009. HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis. Tissue Antigens 74, 17–21. Steenkiste, A., Valdes, A.M., Feolo, M., Hoffman, D., Concannon, P., Noble, J., Schoch, G., Hansen, J., Helmberg, W., Dorman, J.S., Thomson, G., Pugliese, A., 2007. 14th
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International HLA and Immunogenetics Workshop: report on the HLA component of type 1 diabetes. Tissue Antigens 69 (Suppl 1), 214–225. Stewart, G.J., Teutsch, S.M., Castle, M., Heard, R.N., Bennetts, B.H., 1997. HLA-DR, -DQA1 and -DQB1 associations in Australian multiple sclerosis patients. Eur. J. Immunogenet. 24, 81–92. Svejgaard, A., 2008. The immunogenetics of multiple sclerosis. Immunogenetics 60, 275–286. Watts, C., 2004. The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nat. Immunol. 5, 685–692. Welborn, T.A., 1998. The Besselton Study: Mapping Population Health. Cardiovascular and Respiratory Disease Risk Factors in Busselton, Australia. Australiasian Medical Publishing Company, Sydney, pp. 92–98. Wu, J.S., Zhang, M.N., Carroll, W.M., Kermode, A.G., 2008. Characterisation of the spectrum of demyelinating disease in Western Australia. J. Neurol. Neurosurg. Psychiatry 79, 1022–1026. Zivadinov, R., Uxa, L., Zacchi, T., Nasuelli, D., Ukmar, M., Furlan, C., Pozzi-Mucelli, R., Tommasi, M.A., Locatelli, L., Ulivi, S., Bratina, A., Bosco, A., Grop, A., Cazzato, G., Zorzon, M., 2003. HLA genotypes and disease severity assessed by magnetic resonance imaging findings in patients with multiple sclerosis. J. Neurol. 250, 1099–1106.
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
HLA-DRB1 allele heterogeneity influences multiple sclerosis severity as well as risk in Western Australia Jing-Shan Wu a, Ian James c, Wei Qiu a,e, Alison Castley b, Frank T. Christiansen b,d, William M. Carroll a, Frank L. Mastaglia a, Allan G. Kermode a,⁎ a Centre for Neuromuscular and Neurological Disorders, University of Western Australia; Department of Neurology, Sir Charles Gairdner Hospital, Queen Elizabeth II Medical Centre, Perth, Western Australia, Australia b Department of Clinical Immunology and Immunogenetics, PathWest Royal Perth Hospital, Perth, Western Australia, Australia c Centre for Clinical Immunology and Biomedical Statistics, Murdoch University and Royal Perth Hospital, Perth, Western Australia, Australia d School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Western Australia, Australia e Department of Neurology, the Third Affiliated Hospital of Sun Yet-sen University, Guangzhou, China
a r t i c l e
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Article history: Received 25 September 2009 Received in revised form 17 November 2009 Accepted 20 November 2009 Keywords: Multiple sclerosis Human Leukocyte Antigen (HLA) Susceptibility Multiple Sclerosis Severity Score
a b s t r a c t Susceptibility to multiple sclerosis (MS) has been consistently associated with the Human Leukocyte Antigen (HLA)-DRB1⁎1501 genotype, however effects on disease severity and clinical outcome have varied in different populations. We present the results of a high-resolution HLA-DRB1 genotyping and genotype–phenotype correlation study in a large West Australian MS cohort. Our findings indicate that in this population, which is of largely Anglo-Celtic and Northern European origin, HLA-DRB1⁎1501 is not only a strong determinant of disease risk but may also be associated with disease severity as measured by the Multiple Sclerosis Severity Score (MSSS), with the MSSS increasing by an estimated 0.51 per DRB1⁎1501 allele. We also found evidence that the HLA-DRB1⁎1201 allele is associated with less severe disease. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a heterogeneous demyelinating disease whose pathogenesis is thought to involve a complex interaction between genetic factors, which are likely to vary in different populations and ethnic groups, and environmental influences whose importance varies in different geographic locations. Genetic susceptibility is strongly associated with allelic variations at the Human Leukocyte Antigen (HLA)-DRB1 gene locus (Hafler et al., 2007; Lincoln et al., 2005). Among the over 600 HLA-DRB1 alleles sequenced to date, the dominant association is with the HLA-DRB1⁎1501 allele which is found in over 50% of MS cases in all Caucasian populations so far studied (Schmidt et al., 2007) and which is more likely to be transmitted through the maternal line (Ramagopalan et al., 2008b). Studies in Europe and North America have shown that HLA-DRB1⁎1501 increases the risk of developing MS in a dose-dependent manner with homozygotes having several times higher risk than heterozygotes (Barcellos et al., 2003). As well as its effect on MS susceptibility, DRB1⁎1501 has also been found to be associated with a younger age of onset (Hensiek et al., 2002), a greater
⁎ Corresponding author. Australian Neuromuscular Research Institute, Sir Charles Gairdner Hospital; Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Queen Elizabeth II Medical Centre, 6009, Perth, WA, Australia. Tel.: +61 8 93881865; fax: +61 8 93882149. E-mail address: [email protected] (A.G. Kermode). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.11.015
number of brain MRI lesions (Hauser et al., 2000), and a worse outcome (Barcellos et al., 2003; Okuda et al., 2009) in some populations. However the findings with regard to clinical course (Hillert et al., 1992; Olerup et al., 1989) and disease outcome have been inconsistent (Barcellos et al., 2006; Ramagopalan et al., 2008a; Runmarker et al., 1994). Disease susceptibility in HLA-DRB1⁎1501 carriers has recently also been shown to be influenced by synergistic interactions with the alternate parental (in trans) allele at the DRB1 locus (Dyment et al., 2005). Thus, in a study of a large MS cohort DRB1⁎1501/⁎0801 heterozygosity was found to confer a higher risk, comparable to that of DRB1⁎1501 homozygotes, while carriers of the DRB1⁎15/14 genotype had a lower disease risk (Barcellos et al., 2006). Such epistatic interactions may affect the disease phenotype and outcome as well as susceptibility. For example, in a pooled Canadian–Sardinian MS cohort the DRB1⁎01 allele was shown to confer a milder disease outcome when carried in combination with DRB1⁎15 (DeLuca et al., 2007). It has been proposed that the genetic influences on disease susceptibility and outcome may be intrinsically linked (Ramagopalan et al., 2008a). Additional studies in other populations may be helpful to investigate this association further. In the present study high-resolution HLA-DRB1 genotyping was performed in a cohort of 495 well-characterized West Australian MS patients from the Perth Demyelinating Diseases Database (PDDD) (Wu et al., 2008), and the influence of allelic heterogeneity on disease severity was analysed. To our knowledge, this is the first such
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comprehensive study on a large MS cohort from the Southern hemisphere. 2. Methods
and at least 5 cases for MSSS analyses. Among the considered alleles only HLA-DRB1⁎1501, DRB1⁎0301 and DRB1⁎0701 had more than 2 homozygous cases (39, 14 and 5, respectively) and analyses of dose effects and homozygosity were restricted to these alleles. The cohort was shown to be in Hardy–Weinberg equilibrium.
2.1. Research participants 3. Results A total of 504 consecutive patients from the PDDD who were enrolled during the period from November 2006 to December 2008 were reviewed. The original country of birth of the patient and the patient's parents were recorded. Nine patients were non-Caucasian and were excluded from the study. Among the remaining 495 subjects, 466 were diagnosed as clinically definite or probable MS according to the Poser criteria (Poser et al., 1983) or MS using the modified McDonald criteria (Compston et al., 2001); 425 were classified as relapsing–remitting MS (RRMS) (including secondaryprogressive) and 41 as primary-progressive MS (PPMS). The control group comprised 189 Caucasian individuals from the Busselton Community Health Study (Welborn, 1998). The protocol was approved by the Sir Charles Gairdner Hospital Human Research Ethics Committee, and informed consent was obtained from all participants.
3.1. Demographic and clinical data The female-to-male ratio in the study cohort was 3.2:1. The average age of onset was 35.3 years and the mean disease duration was 13.2 years (0–58 years). EDSS and MSSS scores as recorded at the time of the last clinical assessment were obtained in 434 (93.1%) and 431 (92.5%) patients, respectively. The mean EDSS score was 3.5 ± 2.4 and the mean MSSS was 4.1 ± 2.8. The MSSS was strongly associated with age-at-onset (p = 0.0004) and gender (p = 0.002), and was higher in males than in females (4.8 ± 2.7 vs 3.8 ± 2.8, p = 0.001). The association with age-at-onset remains strong (p = 0.0004) even after adjustment for gender and the potential confounding (but nonsignificant) effect of disease duration, with an estimated increase of 0.05/year of age.
2.2. HLA-DRB1 typing 3.2. HLA-DRB1 alleles and MS severity High-resolution 4-digit HLA-DRB1 genotyping was performed in the Department of Clinical Immunology and Immunogenetics PathWest, Royal Perth Hospital by DNA sequencing. The HLA-DRB1 typing was performed using a previously reported method (Sayer et al., 2001) with the following modifications. The DNA was extracted from whole blood using the QIAGEN BioRobot M48 machine using Magattract DNA Blood Mini M48 kit (192). The PCR was performed in a 30 μl volume and purified using Agencourt® AMPure® kits PCR purification system. Automated sequencing was carried out using ABI Big Dye Terminator chemistry on ABI Prism 3730 and 3730xl Genetic Analysers and HLA-DRB1 analysis was carried out using ASSIGNV4.0.1.36 (Conexio Genomics). 2.3. Clinical and laboratory data Patients were assessed clinically by two neurologists in the MS clinic (AGK and WMC) at the time of collecting blood samples. Data recorded included: gender, family history of MS, age-at-clinical onset, disease duration, clinical course, Extended Disability Status Scale (EDSS) score at last visit, Global MS Severity Score (MSSS) (Roxburgh et al., 2005), visual evoked potential (VEP) and MRI findings on the most recent studies and results of cerebrospinal fluid studies. The MSSS scores were calculated based on individual's EDSS adjusted for disease duration using the Global MSSS table (Roxburgh et al., 2005). 2.4. Statistical analysis Comparisons of carriage frequencies of alleles between cases and controls were carried out by case–control logistic regressions for overall multivariable analyses and Fisher exact tests for individual alleles. Comparisons of EDSS and MSSS scores across groups and in multivariable analyses were carried out via linear regression. Analyses were carried out with and without adjustment for gender with consistent results. Overall analyses for each outcome based on the multivariable models are presented along with the univariate analyses for each allele separately. p-values b 0.05 were considered statistically significant. Although the individual tests are not independent, those results withstanding crude Bonferroni adjustment are indicated where appropriate. A total of 34 different HLA-DRB1 alleles were detected in this study cohort. To avoid potential artifacts due to small samples we considered only those alleles carried by at least 5 individuals in the combined samples for case–control comparisons
The carriage frequencies of 25 HLA-DRB1 alleles in cases and controls and the associations of the 21 alleles satisfying our inclusion criteria with MSSS are shown in Table 1. The HLA-DRB1⁎1501 allele was strongly associated with disease risk in both univariate and multivariable analyses, and carriage of this allele was also marginally associated with a higher mean MSSS compared with non-carriers (4.3 vs 3.8, p = 0.04) (Table 1), though this association does not withstand multiple comparison correction. Gender stratification showed no
Table 1 Associations of HLA-DRB1 alleles with MSSS and carriage frequencies in the MS and control groups of subjects. HLADRB1
Case carriage Control carriage Odds p-valuea MSSS mean p-valueb frequency% (n) frequency% (n) ratio difference
⁎0101 ⁎0102 ⁎0103 ⁎0301 ⁎0401 ⁎0402 ⁎0403 ⁎0404 ⁎0405 ⁎0407 ⁎0701 ⁎0801 ⁎0901 ⁎1001 ⁎1101 ⁎1103 ⁎1104 ⁎1201 ⁎1301 ⁎1302 ⁎1303 ⁎1401 ⁎1501 ⁎1502 ⁎1601
11.8% (55) 1.5% (7) 4.7% (22) 23.6% (110) 11.2% (52) 1.3% (6) 1.5% (7) 8.4% (39) 1.3% (6) 0.4% (2) 18.0% (84) 5.8% (27) 1.1% (5) 1.5% (7) 6.0% (28) 0.9% (4) 4.3% (20) 2.6% (12) 7.5% (35) 5.4% (25) 3.9% (18) 2.6% (12) 54.5% (254) 0.1% (2) 3.2% (15)
18.0% (34) 2.7% (5) 2.1% (4) 22.8% (43) 19.1% (36) 2.1% (4) 1.1% (2) 8.5% (16) 0.0% (0) 3.2% (6) 29.6% (56) 3.7% (7) 4.8% (9) 1.1% (2) 12.7% (24) 3.7% (7) 1.1% (2) 4.8% (9) 10.6% (20) 10.1% (19) 1.1% (2) 3.2% (6) 19.6% (37) 2.1% (4) 3.2% (6)
0.61 0.56 2.29 1.05 0.53 0.60 1.43 0.99 inf 0.13 0.52 1.60 0.22 1.43 0.44 0.23 4.19 0.53 0.69 0.51 3.76 0.81 4.92 0.20 1.01
0.04 ns ns ns 0.01 ns ns ns ns 0.01 0.0015c ns 0.01 ns 0.01 0.02 0.05 0.15 ns 0.04 0.08 ns 10− 14c ns ns
− 0.23 − 0.40 − 0.75 − 0.20 − 0.76 0.21 0.03 − 0.19 − − 0.20 0.77 1.77 − 1.25 0.82 − 0.74 − 2.34 − 0.65 − 0.78 0.40 − 0.70 0.56 − 0.29
ns ns ns ns 0.1 ns ns ns − − ns 0.2 ns ns ns − ns 0.003 ns 0.15 ns ns 0.04 − ns
inf: unable to estimate OR. Alleles significantly associated with MSSS were showed in bold. a When HLA-DRB1 allele carriage frequencies were compared between MS cases and healthy controls. b When HLA-DRB1 allele associated mean MSSS were compared between carriers and non-carriers. c p-values remain significant after Bonferroni correction.
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significant difference in the HLA-DRB1⁎1501 MSSS effect for female or male carriers of DRB1⁎1501 (p = 0.6). Of the other alleles analysed the only correlation with MSSS was with HLA-DRB1⁎1201 which was associated with a lower mean MSSS (− 2.3, p = 0.003). This HLADRB1⁎1201 effect remained strong (p = 0.005) even in the multivariable analysis adjusting for the large number of non-significant other alleles, and is close to significant after the crude Bonferroni correction (p = 0.06). 3.3. Dose effect of HLA-DRB1⁎1501 We analysed the HLA-DRB1⁎1501 dose effect for association with disease risk and severity. While numbers were insufficient to distinguish statistically between any effect being dose-dependent rather than simply carriage of DRB1⁎1501, we noted an approximately linear trend in dose effect for both risk (as measured by the OR) and severity (Fig. 1A and B). The MSSS in HLA-DRB1⁎1501 carriers increased by an estimated 0.51 per allele (p = 0.016 uncorrected). 3.4. Other HLA-DRB1 allele combinations Of the other 15 allele combinations analysed, HLA-DRB1⁎1501/0801 had the highest estimated disease risk (OR 8.7, though with small samples this just fails to reach significance, p = 0.07), and was also associated with the highest estimated mean MSSS (5.3 ± 2.5, n = 10), but was not significant when compared with other genotypes
Fig. 1. Dose effect of HLA-DRB1⁎1501 on MS susceptibility and MSSS. A. Carriage of two copies of HLA-DRB1⁎1501 allele conferred a higher estimated disease risk (odds ratio OR = 9.3 relative to no copies) than one copy (OR = 4.5) when compared with healthy controls. B. The MSSS in HLA-DRB1⁎1501 carriers increased by an estimated 0.51 per allele (p = 0.016), with a possible linear dose effect.
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(p = 0.08) (Fig. 2). Although the association between MSSS and other genotypes was not significant, Fig. 2 shows the gradient for the different HLA-DRB1⁎1501 genotypes compared with nonHLA-DRB1⁎1501genotypes. 4. Discussion While the strong genetic association between the HLA-DRB1 genotype and MS susceptibility has been demonstrated consistently across populations, the association with disease severity and outcome has remained more controversial (Barcellos et al., 2003; Barcellos et al., 2006; Ramagopalan et al., 2008a) as environmental and lifestyle factors may also have important effects on the natural history of the disease. The State of Western Australia is ideally suited for genotype– phenotype studies of this type as it has a population of ∼ 2.2 million which is largely of Anglo-Celtic and Northern European origin and is relatively stable. Studies in such geographically isolated and genetically relatively uniform populations may have certain advantages in detecting genetic contributions to disease phenotype and outcome (Hammond et al., 1988) when compared with larger multi-national studies of more heterogeneous patient cohorts. In the present study cohort, nine out of the 504 MS cases (1.8%) were non-Caucasian and were excluded from the study. This figure is comparable to the figure of 3% for outliers in a recently published eigenvector analysis of a European–Australian population sample (McEvoy et al., 2009). In the present study we performed high-resolution (4-point) HLA-DRB1 genotyping to investigate the effects of individual alleles and allele combinations at the HLA-DRB1 locus on disease severity. High-resolution typing allows more reliable definition of allelic variants with different functions which may remain ambiguous with 2-point typing (e.g. the sub-alleles of HLA-DR04 and -DR11) (Listgarten et al., 2008; Svejgaard, 2008). In this population we found that HLA-DRB1⁎1501 is not only the dominant DRB1 allele associated with disease risk as reported in previous Australian studies (Stankovich et al., 2009; Stewart et al., 1997), but may also make an apparent dose-dependent contribution to disease severity, albeit weaker than its contribution to risk. The only other allele which was found to be associated with disease severity was the minor allele HLA-DRB1⁎1201. It is of interest that in previous studies using 2-point genotyping HLA-DRB1⁎12 was found to be associated with lower risk (Ballerini et al., 2004; Masterman et al., 2000),
Fig. 2. Association between HLA-DRB1⁎1501 genotype and MSSS. The MSSS was significantly higher in HLA-DRB1⁎1501/1501 carriers when compared with nonDRB1⁎1501 genotypes (XX/XX) (mean MSSS 4.8 vs 3.8, p= 0.03); HLA-DRB1⁎1501/0801 carriers had the highest mean MSSS of 5.3 but this was not significant when compared with HLA-DRB1⁎XX/XX (p = 0.08).
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whereas in another Italian study HLA-DR1⁎12 was associated with more severe MRI changes (Zivadinov et al., 2003). In the present study HLA-DRB1⁎1201 was found to be associated with a lower MSSS. Our findings are consistent with the notion that epistatic interactions between HLA-DRB1⁎1501 and the other in trans alleles at the DRB1 locus may have a modifying effect on disease severity. Our estimated severity gradient for the various HLA-DRB1⁎1501 genotypes is comparable to that previously found for disease risk in European-derived populations in the Northern Hemisphere (Barcellos et al., 2006; Ramagopalan and Ebers, 2009). These findings support the concept that allelic interactions at the HLA-DRB1 locus may have modifying effects both on MS risk and severity. There is also evidence for similar disease-modifying effects of HLA-DRB1 allele interactions in other autoimmune diseases such as type 1 diabetes mellitus (Price et al., 1999; Steenkiste et al., 2007) and sporadic inclusion body myositis (Mastaglia et al., 2009). The biological mechanisms which underlie these effects remain speculative. It is well known that the proteins encoded by the HLA-DR genes bind antigenic peptides for presentation to CD4+ T lymphocytes (Jones et al., 2006; Watts, 2004). Polymorphic variants of HLA-DRB1 may therefore have differential binding properties for disease-related peptides based upon their amino acid sequences and may thereby modify the risk of inducing the disease as well as its severity and clinical course. As the two alleles at the DRB1 locus are co-dominant and are both expressed, the proteins encoded may present different antigenic epitopes to the immune system. Alternatively, one allele may present a disease-associated epitope and the other could have other effects on the immune response by acting through cytokine networks. However, determining the influence of genetic factors on disease severity and outcome is likely to be more complicated as gene–environment interactions may also play a part. For example, vitamin D can regulate the expression of HLA-DRB1⁎1501 (Ramagopalan et al., 2009), and such epigenetic interactions could potentially affect disease expression. This could also explain our observation of a strong association of HLA-DRB1⁎1501 with disease risk but much weaker association with disease severity. In summary, the present findings indicate that in this MS population allelic heterogeneity at the HLA-DRB1 locus may have modifying effects on disease severity as well as susceptibility. Similar studies in other populations would be helpful in determining the contribution of genetic factors to disease severity and outcome. Funding The National Health and Medical Research Council of Australia and Multiple Sclerosis Research Australia provided a Training Scholarship to Dr Jing-Shan Wu. Dr. Wei Qiu was supported by an Australian Endeavour International Postgraduate Research Scholarship (EIPRS) and Postgraduate Award from The University of Western Australia. Acknowledgements Steve Pummer and the staff at the Department of Clinical Immunology and Immunogenetics, PathWest, Royal Perth Hospital kindly assisted with sample collection and the HLA-DRB1 genotyping. References Ballerini, C., Guerini, F.R., Rombola, G., Rosati, E., Massacesi, L., Ferrante, P., Caputo, D., Talamanca, L.F., Naldi, P., Liguori, M., Alizadeh, M., Momigliano-Richiardi, P., D'Alfonso, S., 2004. HLA-multiple sclerosis association in continental Italy and correlation with disease prevalence in Europe. J. Neuroimmunol. 150, 178–185. Barcellos, L.F., Oksenberg, J.R., Begovich, A.B., Martin, E.R., Schmidt, S., Vittinghoff, E., Goodin, D.S., Pelletier, D., Lincoln, R.R., Bucher, P., Swerdlin, A., Pericak-Vance, M.A., Haines, J.L., Hauser, S.L., 2003. HLA-DR2 dose effect on susceptibility to multiple sclerosis and influence on disease course. Am. J. Hum. Genet. 72, 710–716.
Barcellos, L.F., Sawcer, S., Ramsay, P.P., Baranzini, S.E., Thomson, G., Briggs, F., Cree, B.C., Begovich, A.B., Villoslada, P., Montalban, X., Uccelli, A., Savettieri, G., Lincoln, R.R., DeLoa, C., Haines, J.L., Pericak-Vance, M.A., Compston, A., Hauser, S.L., Oksenberg, J.R., 2006. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum. Mol. Genet. 15, 2813–2824. Compston, A., Edan, G., Goodkin, D., Hartung, H.P., Lublin, F.D., McFarland, H.F., Paty, D.W., Polman, C.H., Reingold, S.C., Sandberg-Wollheim, M., Sibley, W., Thompson, A., van den Noort, S., Weinshenker, B.Y., Wolinsky, J.S., McDonald, W.I., 2001. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann. Neurol. 50, 121–127. DeLuca, G.C., Ramagopalan, S.V., Herrera, B.M., Dyment, D.A., Lincoln, M.R., Montpetit, A., Pugliatti, M., Barnardo, M.C., Risch, N.J., Sadovnick, A.D., Chao, M., Sotgiu, S., Hudson, T.J., Ebers, G.C., 2007. An extremes of outcome strategy provides evidence that multiple sclerosis severity is determined by alleles at the HLA-DRB1 locus. Proc. Natl. Acad. Sci. U. S. A. 104, 20896–20901. Dyment, D.A., Herrera, B.M., Cader, M.Z., Willer, C.J., Lincoln, M.R., Sadovnick, A.D., Risch, N., Ebers, G.C., 2005. Complex interactions among MHC haplotypes in multiple sclerosis: susceptibility and resistance. Hum. Mol. Genet. 14, 2019–2026. Hafler, D.A., Compston, A., Sawcer, S., Lander, E.S., Daly, M.J., De Jager, P.L., de Bakker, P.I., Gabriel, S.B., Mirel, D.B., Ivinson, A.J., Pericak-Vance, M.A., Gregory, S.G., Rioux, J.D., McCauley, J.L., Haines, J.L., Barcellos, L.F., Cree, B., Oksenberg, J.R., Hauser, S.L., 2007. Risk alleles for multiple sclerosis identified by a genomewide study. N. Engl. J. Med. 357, 851–862. Hammond, S.R., McLeod, J.G., Millingen, K.S., Stewart-Wynne, E.G., English, D., Holland, J.T., McCall, M.G., 1988. The epidemiology of multiple sclerosis in three Australian cities: Perth, Newcastle and Hobart. Brain 111 (Pt 1), 1–25. Hauser, S.L., Oksenberg, J.R., Lincoln, R., Garovoy, J., Beck, R.W., Cole, S.R., Moke, P.S., Kip, K.E., Gal, R.L., Long, D.T., 2000. Interaction between HLA-DR2 and abnormal brain MRI in optic neuritis and early MS. Optic Neuritis Study Group. Neurology 54, 1859–1861. Hensiek, A.E., Sawcer, S.J., Feakes, R., Deans, J., Mander, A., Akesson, E., Roxburgh, R., Coraddu, F., Smith, S., Compston, D.A., 2002. HLA-DR 15 is associated with female sex and younger age at diagnosis in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 72, 184–187. Hillert, J., Gronning, M., Nyland, H., Link, H., Olerup, O., 1992. An immunogenetic heterogeneity in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 55, 887–890. Jones, E.Y., Fugger, L., Strominger, J.L., Siebold, C., 2006. MHC class II proteins and disease: a structural perspective. Nat. Rev. Immunol. 6, 271–282. Lincoln, M.R., Montpetit, A., Cader, M.Z., Saarela, J., Dyment, D.A., Tiislar, M., Ferretti, V., Tienari, P.J., Sadovnick, A.D., Peltonen, L., Ebers, G.C., Hudson, T.J., 2005. A predominant role for the HLA class II region in the association of the MHC region with multiple sclerosis. Nat. Genet. 37, 1108–1112. Listgarten, J., Brumme, Z., Kadie, C., Xiaojiang, G., Walker, B., Carrington, M., Goulder, P., Heckerman, D., 2008. Statistical resolution of ambiguous HLA typing data. PLoS Comput. Biol. 4, e1000016. Mastaglia, F.L., Needham, M., Scott, A., James, I., Zilko, P., Day, T., Kiers, L., Corbett, A., Witt, C.S., Allcock, R., Laing, N., Garlepp, M., Christensen, F.T., 2009. Sporadic inclusion body myositis: HLA-DRB1 allele interactions influence disease risk and clinical phenotype. Neuromuscul. Disord. Neuromuscular Disorders 19, 763–765. Masterman, T., Ligers, A., Olsson, T., Andersson, M., Olerup, O., Hillert, J., 2000. HLA-DR15 is associated with lower age at onset in multiple sclerosis. Ann. Neurol. 48, 211–219. McEvoy, B.P., Montgomery, G.W., McRae, A.F., Ripatti, S., Perola, M., Spector, T.D., Cherkas, L., Ahmadi, K.R., Boomsma, D., Willemsen, G., Hottenga, J.J., Pedersen, N.L., Magnusson, P.K., Kyvik, K.O., Christensen, K., Kaprio, J., Heikkila, K., Palotie, A., Widen, E., Muilu, J., Syvanen, A.C., Liljedahl, U., Hardiman, O., Cronin, S., Peltonen, L., Martin, N.G., Visscher, P.M., 2009. Geographical structure and differential natural selection among North European populations. Genome Res. 19, 804–814. Okuda, D.T., Srinivasan, R., Oksenberg, J.R., Goodin, D.S., Baranzini, S.E., Beheshtian, A., Waubant, E., Zamvil, S.S., Leppert, D., Qualley, P., Lincoln, R., Gomez, R., Caillier, S., George, M., Wang, J., Nelson, S.J., Cree, B.A., Hauser, S.L., Pelletier, D., 2009. GenotypePhenotype correlations in multiple sclerosis: HLA genes influence disease severity inferred by 1HMR spectroscopy and MRI measures. Brain 132, 250–259. Olerup, O., Hillert, J., Fredrikson, S., Olsson, T., Kam-Hansen, S., Moller, E., Carlsson, B., Wallin, J., 1989. Primarily chronic progressive and relapsing/remitting multiple sclerosis: two immunogenetically distinct disease entities. Proc. Natl. Acad. Sci. U. S. A. 86, 7113–7117. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H., Tourtellotte, W.W., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227–231. Price, P., Witt, C., Allcock, R., Sayer, D., Garlepp, M., Kok, C.C., French, M., Mallal, S., Christiansen, F., 1999. The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol. Rev. 167, 257–274. Ramagopalan, S.V., Deluca, G.C., Degenhardt, A., Ebers, G.C., 2008a. The genetics of clinical outcome in multiple sclerosis. J. Neuroimmunol. 201–202, 183–199. Ramagopalan, S.V., Herrera, B.M., Bell, J.T., Dyment, D.A., Deluca, G.C., Lincoln, M.R., Orton, S.M., Chao, M.J., Sadovnick, A.D., Ebers, G.C., 2008b. Parental transmission of HLA-DRB1⁎15 in multiple sclerosis. Hum. Genet. 122, 661–663. Ramagopalan, S.V., Ebers, G.C., 2009. Epistasis: multiple sclerosis and the major histocompatibility complex. Neurology 72, 566–567. Ramagopalan, S.V., Maugeri, N.J., Handunnetthi, L., Lincoln, M.R., Orton, S.M., Dyment, D.A., Deluca, G.C., Herrera, B.M., Chao, M.J., Sadovnick, A.D., Ebers, G.C., Knight, J.C., 2009. Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1⁎1501 is regulated by vitamin D. PLoS Genet. 5, e1000369. Roxburgh, R.H., Seaman, S.R., Masterman, T., Hensiek, A.E., Sawcer, S.J., Vukusic, S., Achiti, I., Confavreux, C., Coustans, M., le Page, E., Edan, G., McDonnell, G.V., Hawkins, S., Trojano, M., Liguori, M., Cocco, E., Marrosu, M.G., Tesser, F., Leone, M.A.,
J.-S. Wu et al. / Journal of Neuroimmunology 219 (2010) 109–113 Weber, A., Zipp, F., Miterski, B., Epplen, J.T., Oturai, A., Sorensen, P.S., Celius, E.G., Lara, N.T., Montalban, X., Villoslada, P., Silva, A.M., Marta, M., Leite, I., Dubois, B., Rubio, J., Butzkueven, H., Kilpatrick, T., Mycko, M.P., Selmaj, K.W., Rio, M.E., Sa, M., Salemi, G., Savettieri, G., Hillert, J., Compston, D.A., 2005. Multiple Sclerosis Severity Score: using disability and disease duration to rate disease severity. Neurology 64, 1144–1151. Runmarker, B., Martinsson, T., Wahlstrom, J., Andersen, O., 1994. HLA and prognosis in multiple sclerosis. J. Neurol. 241, 385–390. Sayer, D., Whidborne, R., Brestovac, B., Trimboli, F., Witt, C., Christiansen, F., 2001. HLADRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors. Tissue Antigens 57, 46–54. Schmidt, H., Williamson, D., Ashley-Koch, A., 2007. HLA-DR15 haplotype and multiple sclerosis: a HuGE review. Am. J. Epidemiol. 165, 1097–1109. Stankovich, J., Butzkueven, H., Marriott, M., Chapman, C., Tubridy, N., Tait, B.D., Varney, M.D., Taylor, B.V., Foote, S.J., Kilpatrick, T.J., Rubio, J.P., 2009. HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis. Tissue Antigens 74, 17–21. Steenkiste, A., Valdes, A.M., Feolo, M., Hoffman, D., Concannon, P., Noble, J., Schoch, G., Hansen, J., Helmberg, W., Dorman, J.S., Thomson, G., Pugliese, A., 2007. 14th
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International HLA and Immunogenetics Workshop: report on the HLA component of type 1 diabetes. Tissue Antigens 69 (Suppl 1), 214–225. Stewart, G.J., Teutsch, S.M., Castle, M., Heard, R.N., Bennetts, B.H., 1997. HLA-DR, -DQA1 and -DQB1 associations in Australian multiple sclerosis patients. Eur. J. Immunogenet. 24, 81–92. Svejgaard, A., 2008. The immunogenetics of multiple sclerosis. Immunogenetics 60, 275–286. Watts, C., 2004. The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nat. Immunol. 5, 685–692. Welborn, T.A., 1998. The Besselton Study: Mapping Population Health. Cardiovascular and Respiratory Disease Risk Factors in Busselton, Australia. Australiasian Medical Publishing Company, Sydney, pp. 92–98. Wu, J.S., Zhang, M.N., Carroll, W.M., Kermode, A.G., 2008. Characterisation of the spectrum of demyelinating disease in Western Australia. J. Neurol. Neurosurg. Psychiatry 79, 1022–1026. Zivadinov, R., Uxa, L., Zacchi, T., Nasuelli, D., Ukmar, M., Furlan, C., Pozzi-Mucelli, R., Tommasi, M.A., Locatelli, L., Ulivi, S., Bratina, A., Bosco, A., Grop, A., Cazzato, G., Zorzon, M., 2003. HLA genotypes and disease severity assessed by magnetic resonance imaging findings in patients with multiple sclerosis. J. Neurol. 250, 1099–1106.