Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on June 1, 2005
Human Molecular Genetics 2005 14(14):2019-2026; doi:10.1093/hmg/ddi206

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 14/14/2019    most recent ddi206v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (56) Request Permissions Google Scholar Articles by Dyment, D. A. Articles by Ebers, G. C. Search for Related Content PubMed PubMed Citation Articles by Dyment, D. A. Articles by Ebers, G. C. Social Bookmarking          What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Complex interactions among MHC haplotypes in multiple sclerosis: susceptibility and resistance

David A. Dyment¹, Blanca M. Herrera¹, M. Zameel Cader¹, Cristen J. Willer¹, Matthew R. Lincoln¹, A. Dessa Sadovnick², Neil Risch³ and George C. Ebers^1,*

¹The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, ²Department of Medical Genetics and Faculty of Medicine, Division of Neurology, University of British Columbia, Vancouver, Canada and ³Department of Genetics, Stanford University, California, USA

^* To whom correspondence should be addressed at: Department of Clinical Neurology, University of Oxford, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK. Tel: +44 1865287659; Fax: +44 1865287533; Email: george.ebers{at}clneuro.oxford.ac.uk

Received March 9, 2005; Revised May 13, 2005; Accepted May 24, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
Mechanisms for observed associations within the major histocompatibility complex (MHC) and autoimmune diseases including multiple sclerosis (MS) remain uncertain. Genotyping of the HLA Class II DRB1 locus in 4347 individuals from 873 multiplex families with MS highlights the genetic complexity of this locus. Excess allele sharing in sibling pair families lacking DRB1*15 and DRB1*17 (58.5% sharing; P=0.012) was comparable to that seen where parents were DRB1*15 positive (62%, P=0.0006). DRB1*17 (P=0.00027) was clearly established as an MS susceptibility allele in addition to DRB1*15 (P<10^–14). DRB1*14 showed striking under-transmission (P=0.000032) to affected offspring newly establishing this allele as a broadly acting resistance factor. Trans interactions were seen in both DRB1*15 and non-DRB1*15 bearing genotype combinations. DRB1*08 was transmitted preferentially with DRB1*15 (P=0.0114) and, in the presence of DRB1*08, the transmission of DRB1*15 was almost invariable (37 transmissions to one non-transmission). DRB1*01 was under-transmitted to offspring in the presence of DRB1*15 (P=0.019). Both DRB1*01 and DRB1*14 haplotypes carry DQA1*01-DQB1*05 alleles, suggesting a common DQ-related mechanism for the protection mediated by these haplotypes. These studies demonstrate that it is the Class II genotype that determines susceptibility and resistance to MS. By analogy with celiac disease and type I diabetes, the pattern of susceptibility strongly supports an autoimmune aetiology.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
Genetic epidemiological studies overwhelmingly indicate the influence of genes on susceptibility to multiple sclerosis (MS) (1–3). Nevertheless, there has been little progress in the identification of non-MHC susceptibility loci. For decades, the major histocompatibility complex (MHC) has been unambiguously associated with MS susceptibility (4,5). Initial associations were observed with the human leukocyte antigen (HLA) Class I region of the MHC (6,7) and later with polymorphisms in the HLA Class II region (8). Susceptibility has been fine-mapped to an extended HLA Class II haplotype DQA1*0102 DQB1*0602 DRB1*1501 DRB5*0101(9) with an estimated relative risk (RR) for this haplotype between 2 and 4 (10). Larger overall locus effects have been reported in relatively small samples using haplotype sharing as a measure (11,12). In larger samples, the same calculation gives a population RR (_MHC) of 1.5, implying that this locus accounts for a modest 14% of the total _sibs (13,14). However, prior calculations based on association with DRB1*1501 do not allow for the complex and genotypic-dependent interactions observed in other complex diseases such as coeliac disease (15), IDDM (16) and narcolepsy (17).

In addition to the association with the initially observed DRB1*1501 haplotype, there have been reports demonstrating the allelic heterogeneity at the DRB1 locus in various Southern European Ethnic groups (18–20). There is also evidence for non-Class II MHC susceptibility loci (14,21,22) and candidates have included TNF (23) and HLA-A (24). There have been sufficient data to dispel the notion that MHC-related susceptibility reflects the role of the HLA Class II allele DRB1*1501 alone.

The degree of polymorphism and linkage disequilibrium in the MHC region necessitate very large sample sizes to adequately examine allelic interactions. Here, we present a well-powered investigation into the complexity of the MHC within samples of affected relative MS pairs of siblings, cousins and aunt/uncle/niece/nephews (AUNN) and in addition, singleton ‘sporadic’ case families. We demonstrate considerable complexity at this locus, finding several features of the MHC common to disorders for which there is more direct evidence of an autoimmune pathogenesis. These data provide some explanation for previous observations and highlight additional challenges for understanding the nature of the MHC association.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
The DRB1*15 association
Affected subjects from each type of multiplex and singleton family (see Materials and Methods) showed a positive association with DRB1*15. In the total sample, the DRB1*15 allele was transmitted 634 times and not transmitted 252 times; ²=164.70, P<10^–14 (Table 1). DRB1*15 was present in 55.6% (990/1781) of the MS patients and the allele frequency was 33%. In normal controls, the allele frequency of DRB1*15 was 12.6%, and 23% (41/178) of the controls were DRB1*15 positive. The RR for the DRB1*15 allele in MS patients was 2.41 (CI 1.84–3.17). The transmission of DRB1*15 to the unaffected offspring was also tested in these families. There was no significant transmission distortion. Eighty DRB1*15 alleles were transmitted and 93 DRB1*15 alleles were not transmitted, ²=0.98, P=0.32. A difference in the transmission of DRB1*15 was not significant between the multiplex (OR=2.5) and the singleton families (OR=2.8), although the number of singleton families was relatively small (n=73).

View this table: [in this window] [in a new window]   Table 1. TDT analysis in the total MS family sample

 
Evidence for other allelic associations at the DRB1 locus
The over-transmission of DRB1*15 confounds the identification of other susceptibility and protective alleles. Therefore, to assess allelic associations other than DRB1*15, transmissions from non-DRB1*15 parents (i.e. X/Y—where X and Y are alleles other than DRB1*15) were examined and are presented in Table 2. This was assessed in matings where at least one parent was X/Y; the other parent could carry zero, one or two copies of DRB1*15 or could be of ‘unknown’ genotype. The most prominent associations in the combined sample are with the DRB1*17 allele (P=0.0030) and with the protective DRB1*14 allele (P=0.000055). A conservative Bonferroni correction for the 13 comparisons was applied and both DRB1*17 (P_c=0.039) and DRB1*14 (P_c=0.00072) associations remained significant. Alleles 11 and 12 were also under-transmitted and allele 8 was over-transmitted in the combined sample, but failed statistical significance upon stringent correction for multiple testing.

View this table: [in this window] [in a new window]   Table 2. Transmission of DRB1 alleles from non-DRB1*15 parents ranked by odds ratio

 
To test whether the transmissions from DRB1*15 negative parents were consistent with other families, we counted transmission of alleles from DRB1*15 heterozygous parents. The known association of DRB1*15 was subtracted from the total, in order to control for the known under-transmission of the other non-DRB1*15 alleles (Table 3). No transmissions were formally significant; however, the odds ratios of DRB1*14 (OR=0.46) and DRB1*17 (OR=1.2) were comparable with the odds ratios observed in Table 2. The same is observed for DRB1*11 and DRB1*12, both are again under-transmitted and consistent with the odds ratios in Table 2.

View this table: [in this window] [in a new window]   Table 3. Transmission from DRB1*15 heterozygous parents

 
An interaction with DRB1*15
Transmission to MS offspring from a non-DRB1*15 bearing parent i.e. X/Y was also assessed, stratified by the presence or absence of DRB1*15. For example, in a mating of an X/Y parent and an X/15 parent, the child could be DRB1*15 positive or negative based upon transmission from the X/15 bearing parent. We then assessed the transmission of alleles from the non-DRB1*15 X/Y parent in both scenarios (Table 4). If there were no interactions between DRB1*15 and other DRB1 alleles, the transmission ratios should be the same for DRB1*15 positive and negative offspring.

View this table: [in this window] [in a new window]   Table 4. Transmission of DRB1 alleles from non-DRB1*15 parents to offspring stratified by the presence or absence of DRB1*15

 
The previously associated alleles DRB1*14 and DRB1*17 were found to be consistently under- and over-represented, respectively, in both DRB1*15 positive and negative children. For a number of other alleles including DRB1*09, *11, *12, *13 and *16, transmission was similar in 15 positive and negative children. For others, this was less clearly the case. Several alleles were dichotomized by stratification into DRB1*15 and non-DRB1*15 positive offspring, for example, DRB1*01. This allele was under-transmitted to affected DRB1*15 positive offspring with an odds ratio of 0.57 (P=0.011), however, when DRB1*15 was not present in the affected offspring, there was no distortion of transmission, suggesting that DRB1*01 is protective only in the presence of DRB1*15 (Table 4).

DRB1*08 showed the opposite trend as it was preferentially transmitted to DRB1*15 positive offspring with an odds ratio of 2.38 (P=0.0067), but was not over-transmitted when DRB1*15 was absent in the affected offspring (OR=1.15); this observation suggests that DRB1*08 is predisposing only in the presence of DRB1*15.

DRB1*10 also showed differences between the DRB1*15 defined groups (P=0.022) in that it was under-transmitted to the DRB1*15 positive offspring with an odds ratio of 0.17, whereas it was over-transmitted to the DRB1*15 negative children with an odds ratio of 2.25.

We hypothesized that the transmission of DRB1*15 from the DRB1*15 heterozygous parents would also be skewed when the children are stratified based upon the presence of DRB1*01 and DRB1*08. Moreover, any transmission differences should expectedly be in the same direction as that observed in Table 4. This is a test of the RR of the allele in the presence of DRB1*15; e.g. for DRB1*01, it is a test of DRB1*15/1 versus DRB1*15/X, where X is not DRB1*01. Transmissions were counted from heterozygous DRB1*15 positive parents to those children who are positive or negative for DRB1*01 or *08 inherited from the other parent (Table 5).

View this table: [in this window] [in a new window]   Table 5. Testing interactions of DRB1*15 with DRB1*01 and *08

 
The findings observed in Table 5 are largely consistent with Table 4; the odds ratio of DRB1*15 transmission from a DRB1*01 negative parent to a DRB1*01 positive child (OR=1.6) is less than the odds ratio for a DRB1*01 negative child (OR=2.5). Although not significant (P=0.08), the trend is ‘as expected’ from the original observation (Table 4). The same findings are observed with DRB1*08 stratified children, DRB1*15 was transmitted with an odds ratio of 2.3 to the non-DRB1*08 bearing children, but when DRB1*08 was present, DRB1*15 was transmitted almost invariably (37 transmissions to one non-transmission).

Evidence for non-DRB1*15 bearing haplotypes linked to MS
We have previously shown increased sharing at the HLA DRB1 locus in MS sibling pairs having both parents DRB1*15 negative (13). For Table 6, allele sharing at DRB1 was assessed in sibling pairs from the total non-HLA DRB1*15-bearing parents. For example, sharing from a DRB1*X/Y mother was included in the analysis even when the father was DRB1*15/15 or DRB1*15/X. This is different from our previous analysis (13) when only families with both parents negative for DRB1*15 were counted.

View this table: [in this window] [in a new window]   Table 6. Sharing of haplotypes inherited from one or both non-DRB1*15-bearing parents in MS sibling pairs

 
The haplotype sharing from the DRB1*X/Y parents from the total sibling pair sample was 58.0% (²=7.68; P=0.0056). As we have shown that DRB1*17 is also positively associated with MS in the Canadian sample, we repeated the analysis with parents who were DRB1*15 and DRB1*17 negative (Table 7). In families with both parents negative for the two susceptibility alleles (n=30), the sharing was 70.5% (²=10.25; P=0.0013). Sharing from all parents lacking DRB1*17 and DRB1*15 was 58.5% (²=6.31; P=0.012).

View this table: [in this window] [in a new window]   Table 7. Sharing of haplotypes inherited from one or both non-DRB1*15 or *17-bearing parents in MS sibling pairs

 
As a comparison, the sharing of DRB1 alleles was also assessed among sibling pairs with parents heterozygous DRB1*15/X. The sharing observed from these heterozygous parents was 62.5% (²=16.1; P=0.0006) and not significantly different than the sharing observed when parents lacked DRB1*15 or DRB1*17.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
The mechanisms by which HLA Class II associations impart risk for most diseases are yet to be defined. However, it is widely believed that the primary association for these putative autoimmune diseases is with alleles at these loci. In the case of MS, the HLA Class II DQA1*0102-DQB1*0602-DRB1*1501-DRB5*0101 haplotypic association provides evidence in support of an autoimmune reaction against myelin-related antigens presented to T-cells in the restricting context of HLA DRB1*1501.

Despite circumstantial evidence in support of this hypothesis, there have been a number of observations suggesting that this interpretation is facile. The data in this study suggest a more complex picture characterized not only by locus and allelic heterogeneity, but also by evidence of interactions that modify the RR to develop MS.

DRB1*15
The well-documented association with HLA DRB1*15 was observed in this sample of 873 Canadian MS families (Table 1). However, the effect of DRB1*15 is modest with an RR of 2.4, with only 55% of MS patients bearing this susceptibility allele (25,26).

Other allelic associations
If DRB1*15 was itself the sole susceptibility allele in the MHC, we should observe no increased sharing in sibling pairs where both parents are DRB1*15 negative. However, we observed 62.7% sharing at the DRB1 locus (13). Our conclusion, based upon the previous finding, was that there are haplotypes or loci other than DRB1*15 contributing to MS risk. We have extended this linkage evidence in the absence of the DRB1*15 bearing haplotypes by examining linkage in sibling pairs with any parent DRB1*15 negative (Table 6). The results show an increased sharing of 58% among the sibling pairs, from all DRB1*15 negative parents, with an mlod=1.7; ²=7.68; P=0.0056. This is similar to the 62% sharing seen in sibling pairs from a DRB1*15/X parent.

To test the hypothesis that allelic heterogeneity may be responsible for this increased sharing, we examined whether other alleles were transmitted preferentially to affected offspring from non-DRB1*15 bearing parents. A number of other alleles were observed to increase and decrease risk of MS. Allele DRB1*17 is significantly associated with MS upon correction for multiple comparisons and is in keeping with previous case–control studies of Northern European MS patients (27,28). This finding is also consistent with findings from non-Northern European patients. Mexican and Sardinian MS patients both have an association with DRB1*17 (18,29). Consistent with this hypothesis is the observation that the Sardinian MS population is also associated with DRB1*15, but it was not originally identified due to the relatively low frequency of DRB1*15 alleles in the population (14). In this Canadian sample, 72% (1276/1781) of patients assessed were positive for DRB1*15 and/or DRB1*17 compared with 48% (85/178) of controls.

In contrast to the HLA*DRB1*15 and HLA DRB1*17 alleles, HLA DRB1*14 was identified as a strongly protective allele with an odds ratio of 0.31. This is as large a relative effect as the odds ratio for DRB1*15 (OR=2.5) for susceptibility and this has not been reported previously. DRB1*11 and DRB1*12 also showed a trend to significance with odds ratios of 0.75 and 0.55, respectively. This trend was consistent with the transmissions of DRB1*11 and DRB1*12 from DRB1*15 heterozygous parents (Table 3). Some alleles appeared to have a ‘neutral’ effect on risk (DRB1*13, *16 and *07); however, there may exist subgroups within the sample that may be associated with MS because some of the effects only emerge upon stratification.

HLA interactions with the DRB1*15-bearing haplotype
The transmission of DRB1 alleles from non-DRB1*15 parents stratified by the presence or absence of the DRB1*15 in the offspring was largely consistent; alleles DRB1*04, *09,*11, *12, *13 and *16 were observed to be equally transmitted between the two groups (Table 4). The same was true for DRB1*14 and DRB1*17, which were consistently under- and over-transmitted between the two groups, respectively.

Another positive trend for association was with the relatively rare allele DRB1*8 (Table 2). However, when the findings were restricted to patients who were DRB1*15 positive, the odds ratio for DRB1*8 increased from 1.64 to 2.39, P=0.0067 (Table 4). The non-DRB1*15 bearing MS offspring showed no transmission distortion of DRB1*8 and the odds ratio was 1.15.

DRB1*01 also showed distinct dimorphic patterns of transmission between the two groups. In the case of DRB1*01, the non-DRB1*15 bearing parents under-transmitted DRB1*01 to DRB1*15 positive offspring (Table 4). DRB1*10 seems to show a similar pattern in that DRB1*10 was over-transmitted to DRB1*15 negative children (OR=2.25) and under-transmitted to DRB1*15 positive children (OR=0.17).

To confirm or refute these findings for haplotypic interactions, we tested the transmission from DRB1*15 positive parents (versus DRB1*15 negative of Table 4) to offspring, in other words, the reciprocal of the previous analysis. The results were remarkably similar (Table 5) with DRB1*15 being relatively under-transmitted to DRB1*01 positive offspring when compared with DRB1*01 negative offspring (²=3.01; P=0.08). DRB1*15 was also preferentially transmitted to DRB1*08 positive offspring when compared with non-DRB1*08 bearing offspring (²=13.21; P=0.0002). Although DRB1*15 also showed similar findings when stratified by the presence or absence of DRB1*10, the sample size was exceedingly small with only three transmissions of DRB1*15 counted to the DRB1*10 positive offspring.

DRB1*01 has previously been implicated as a protective allele but of low magnitude; our odds ratio was 0.86 (30–34). Importantly, the three negatively associated DRB1 alleles (DRB1*01, *14 and *10) have in common the extended DQ haplotype DQA1*01-DQB1*05, suggesting that resistance may be encoded by the DQ genes versus the DRB1 alleles. This DQ haplotype is also protective in narcolepsy and DRB1*14 has been shown to be a protective haplotype in IDDM.

The interactions observed between DRB1 haplotypes suggest that the gene products are interacting to increase or decrease MS risk. The mechanism is unlikely to be operating via antigen presentation by the HLA DRß/HLA DR dimer complex, as the HLA DRA1 gene is relatively invariant. If the DRB1*15 restricted DRB1*01/*08/*10 interactions are true, this would lend further evidence implicating the DQ genes in MS susceptibility. Both the HLA DQA and HLA DQB genes are highly polymorphic, and they are in tight disequilibrium. From a functional perspective, the DQA and DQB alleles interact to form dimers for T-cell presentation. It is plausible that the formation of the HLA DQ and HLA DQ ß heterodimers may provide a molecular mechanism for the haplotypes to interact in trans to increase the MS risk, as has been suggested in IDDM and coeliac disease.

In IDDM, the DRB1*03/*04 heterozygotes have a greater RR when compared with DRB1*03 or *04 homozygotes. This is thought to be the result of trans-encoded HLA DQ genes encoding highly susceptible DQ 1ß1 dimers (16). A similar mechanism is observed in coeliac disease with patients carrying the DQA1*05-DQB1*02 alleles in trans and also in cis, and in narcolepsy, the DQB1*0602 shows increased risk when DQB1*0601, DQB1*0501 or DQA1*01 are present in trans (15). These similarities support the evidence that MS is an autoimmune disorder with strong parallels to IDDM. Although DRB1*1501 is a susceptibility allele in MS and imparts resistance in IDDM, other alleles, as shown in this study, behave similarly in the two conditions. We have shown here that the alleles DRB1*01, *11 and particularly DRB1*14 unexpectedly impart resistance in MS as has been reported for IDDM. With these similarities, it might be expected that MS may share other susceptibility loci with other autoimmune conditions. However, PTPN22 was shown to be associated with IDDM, rheumatoid arthritis, systemic lupus erythematous and Graves disease (35) was not associated to MS (36).

Further insight suggesting HLA DQ involvement in MS was observed in a study of Afro-Brazilian MS patients which showed a positive association for the DQ alleles in the absence of the DRB1*1501 allele (37). Moreover, a recent Sardinian study of over 400 MS patients demonstrated that both DRB1 and DQB1 act independently to increase MS risk (14). In contrast, among African-American MS patients, the DRB1 locus appears to be the susceptible locus rather than the DQ loci (38). If both DR and DQ independently and in combination determine risk, population associations at these loci could favour one or the other depending on allele frequencies and additional haplotypic features. The DQ locus was not genotyped for this investigation, though it would be beneficial for future resolution of the involvement of these loci in MS susceptibility.

Non-Class II MHC susceptibility loci
Although the alleles implicated in MS susceptibility may represent a functional hierarchy related to antigen presentation, it cannot be excluded that they are simply in functional or physical disequilibrium with as yet unidentified loci. The literature contains many reports in support of a non-Class II susceptibility locus (14,21–24). In favour of non-DRB1 susceptibility loci, the observation that haplotype sharing where both parents were DRB1*15 negative is very similar to when parents are positive (Table 6). Furthermore, when we examined sharing from parental haplotypes lacking both DRB1*15 and DRB1*17, the finding was still increased at 58.5% (Table 7). This suggests that not only allelic heterogeneity, but also locus heterogeneity exists at the MHC locus. Recent scans spanning the MHC support this hypothesis (14,21,22).

Complexity and MHC
These results serve to highlight the genetic complexity of the MHC in MS, a putative autoimmune disorder. Although it is possible that the identification of specific susceptibility genes may eliminate much of this, there remain many unresolved features of this region to date which are central to its role in disease. We observed multiple alleles, protective alleles and the effects of allelic interactions. The observations of MHC-linked complexity will be of importance to MS and other autoimmune diseases, where there is also increasing evidence of MHC heterogeneity (39,40). The MHC is a logical target for a high-density single nucleotide polymorphism map in disease (41) and we have completed a high-density map of this region in more than 2000 affected individuals (submitted for publication). The results localize MHC-related susceptibility to the MHC Class II region alone.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
MS patients and their ascertainment
The families were ascertained as part of an ongoing Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS), for which the methodology has been previously described (42). The material consisted of those with sibling pairs (n=442), cousin pairs (n=184), AUNN pairs (n=174) and singleton families (n=73) for a total of 873 families and 1781 affected offspring (Table 8). Families were counted once; for example, if a family included an affected aunt plus two affected nephews (i.e. a sibling pair), the family was counted once as an AUNN relative pair family. The families were of Canadian and Caucasian descent. One-hundred and seventy-eight unaffected and unrelated individuals were also genotyped to serve as a normal control sample. They were also of Canadian and Caucasian descent.

View this table: [in this window] [in a new window]   Table 8. MS families genotyped

 
Genotyping
The genotyping of the HLA DRB1 gene was performed using a low-resolution allele-specific PCR amplification method (43). Twenty-four reactions amplified allelotypes corresponding to alleles DRB1*1, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 as well as amplicons for the DRB3, DRB4 and DRB5 genes. Allele frequencies were estimated from an additional 178 unrelated and unaffected individuals of Caucasian descent.

Four-thousand three-hundred and forty-seven individuals from the 873 families were genotyped at DRB1 locus.

Statistical analysis
Transmission disequilibrium test (TDT) was performed using the sib_tdt program of ASPEX 2.3 analysis package available at (ftp://lahmed.stanford.edu/pub/aspex). The TDT counts the number of times an allele is transmitted to affected offspring from heterozygous parents (44). Parents who were not genotyped were reconstructed whenever possible from the unaffected siblings only. In the case when one parent was unknown, the transmissions were counted only in those instances where both the genotyped parent and the affected offspring were heterozygous for different alleles (e.g. a 7, 15 parent and 7,15 child were not counted), in order to avoid directional bias. The 184 cousin pair families and the 174 AUNN pair families were separated into independent nuclear families for the TDT analysis. Statistical significance was determined empirically with Monte Carlo simulation. The allele labels of heterozygous parents were inverted randomly with probability of 50%, and the TDT ² statistics was calculated for each permutation step. The proportion of simulated values that exceed the observed value is the derived empirical significance level (45).

Non-parametric linkage analyses were performed with the sib_ibd program of Aspex 2.3 package.

The odds ratios and the chi-squared values for the combined data were calculated by Mantel–Haenszel analysis (46).

	MEMBERS OF THE CANADIAN COLLABORATIVE STUDY GROUP

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 
Vancouver, British Columbia: D. Paty, MD, J. Oger, MD, V. Devonshire, MD, S. Hashimoto, MD, J. Hooge, MD, L. Kastrukoff, MD, T. Traboulsee, MD; London, Ontario: G. Rice, MD; M. Kremenchutzky; Calgary, Alberta: L. Metz, MD; Edmonton, Alberta: S. Warren, PhD; Saskatoon, Saskatchewan: W. Hader, MD; Toronto, Ontario: P. O'Connor, MD, T. Grey, MD, M. Hohol MD; Ottawa, Ontario: M. Freedman, MD; Kingston, Ontario: R. Paulseth, MD; Montreal, Quebec: Y. Lapierre, MD (Montreal Neurological Institute), P. Duquette, MD (Hopital Notre Dame); Quebec City, Quebec: J-P. Bouchard, MD; Halifax, Nova Scotia: T. Murray, MD, V. Bhan, MD, C. Maxner, MD; St. John's, Newfoundland: W. Pryse-Phillips, MD, M. Stefanelli, MD.

	ACKNOWLEDGEMENTS

 
We would especially like to thank Holly Armstrong, Beverly Scott, Dr Jan Hillert, Dr Arturs Ligers, Dr Jamie Steckley, Katie Morrison and Angie Shaw for their help with genotyping and data organization. Informed consent was obtained from all subjects and the experiments performed for this investigation comply with current guidelines and ethics. Funding for the Canadian Collaborative Project on Genetic Susceptibility to MS is by the MS Society of Canada Scientific Research Foundation. The MS Society of Canada has funded studentships to D.A.D. and C.J.W. A.D.S. is a Michael Smith Distinguished Scholar.

Conflict of Interest statement. A. Sadovnick is a member of the Speaker's Bureau for Berlex, Biogen, Teva and Serono. No other conflicts of interest were declared.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS MEMBERS OF THE CANADIAN... REFERENCES

 

1. Ebers, G.C., Sadovnick, A.D., Risch, N.J. and the Canadian Collaborative Study Group. (1995) A genetic basis for familial aggregation in multiple sclerosis. Nature, 377, 150–151.[CrossRef][Medline]

2. Sadovnick, A.D., Ebers, G.C., Dyment, D.A., Risch, N.J. and the Canadian Collaborative Group. (1996) A population-based half-sib study of multiple sclerosis. Lancet, 347, 1728–1730.[CrossRef][Web of Science][Medline]

3. Willer, C.J., Dyment, D.A., Risch, N.J., Sadovnick, A.D. and Ebers, G.E. (2003) Twin concordance and sibling recurrence rates in multiple sclerosis: the Canadian Collaborative Study. Proc. Natl Acad. Sci. USA, 100, 12877–12882.[Abstract/Free Full Text]

4. Dyment, D.A., Ebers, G.C. and Sadovnick, A.D. (2004) The genetics of MS. Lancet Neurol., 3, 104–110.[CrossRef][Web of Science][Medline]

5. Herrera, B.M. and Ebers, G.C. (2003) Progress in deciphering the genetics of multiple sclerosis. Curr. Opin. Neurol., 16, 253–258.[CrossRef][Web of Science][Medline]

6. Naito, S., Namerow, N., Mickey, M. and Teraski, P. (1972) Multiple sclerosis: association with HL-A3. Tissue Antigens, 2, 1–4.[Web of Science][Medline]

7. Jersild, C., Svejgaard, A. and Fog, T. (1972) HL-A antigens and multiple sclerosis. Lancet, I, 1240–1241.[CrossRef]

8. Winchester, R.J., Ebers, G., Fu, S.M., Espinosa, L., Zabriskie, J. and Kunkel, H.G. (1975) B-cell alloantigen Ag7a in multiple sclerosis. Lancet, I, 814.

9. Fogdell, A., Hilleret, J., Sachs, C. and Olerup, O. (1995) The multiple sclerosis- and narcolepsy-associated HLA class II haplotype includes DRB5*0101 allele. Tissue Antigens, 46, 333–336.[Web of Science][Medline]

10. Tiwari, J.L. and Terasaki, P.I. (1985) HLA and Disease Associations. Springer-Verlag, New York, NY.

11. Haines, J.L., Terwedow, H.A., Burgess, K., Pericak-Vance, M.A., Rimmler, JB., Martin, E.R., Oksenberg, J.R., Lincoln, R., Zhang, D.Y., Banatao, D.R. et al. (1998) Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. Hum. Mol. Genet., 7, 1229–1234.[Abstract/Free Full Text]

12. Risch, N. (1987) Assessing the role of HLA-linked and unlinked determinants of disease. Am. J. Hum. Genet., 40, 1–14.[Web of Science][Medline]

13. Ligers, A., Dyment, D.A., Willer, C.J., Sadovnick, A.D., Ebers, G., Risch, N., Hillert, J. and Canadian Collaborative Study Groups. (2001) Evidence of linkage with HLA-DR in DRB1*15-negative families with multiple sclerosis. Am. J. Hum. Genet., 69, 900–903.[CrossRef][Web of Science][Medline]

14. Marrosu, M.G., Murru, R., Murru, M.R., Costa, G., Zavarrati, P., Whalen, M., Cocco E., Mancosu, C., Schirru, L., Solla, E. et al. (2001) Dissection of the HLA association with multiple sclerosis in the founder isolated population of Sardinia. Hum. Mol. Genet., 10, 2907–2916.[Abstract/Free Full Text]

15. Margaritte-Jeannin, P., Babron, M.C., Bourgey, M., Louka, A.S., Clot, F., Percopo, S., Coto, I., Hugot, J.P., Ascher, H., Sollid, L.M. et al. (2004) HLA-DQ relative risks for coeliac disease in European populations: a study of the European genetics cluster on coeliac disease. Tissue Antigens, 63, 562–567.[CrossRef][Medline]

16. Koeleman, B.P., Lie, B.A., Undlien, D.E., Dudbridge, F., Thorsby, E., de Vries, R.R., Cucca, F., Roep, B.O., Giphart, M.J. and Todd, J.A. (2004) Genotype effects and epistasis in type 1 diabetes and HLA-DQ trans dimer associations with disease. Genes Immun., 5, 381–388.[CrossRef][Medline]

17. Mignot, E., Lin, L., Rogers, W., Honda, Y., Qin, V., Lin, X., Okun, M., Hohjoh, H., Miki, T., Hsu, S. et al. (2001) Complex HLA-DR and -DQ interactions confer risk of narcolepsy–cataplexy in three ethnic groups. Am. J. Hum. Genet., 68, 686–699.[CrossRef][Web of Science][Medline]

18. Marrosu, M.G., Murru, M.R., Costa, G., Cucca, F., Sotgiu, S., Rosati, G. and Muntoni, F. (1997) Multiple sclerosis in Sardinia is associated and in linkage-disequilibrium with HLA-DR3 and -DR4 alleles. Am. J. Hum. Genet., 61, 454–457.[Web of Science][Medline]

19. Marrosu, M.G., Fadda E., Mancosu, C., Lai, M., Cocco, E. and Pugliati, M. (2000) The contribution of HLA to multiple sclerosis susceptibility in Sardinian affected sibling pairs. Ann. Neurol., 47, 411–412.[CrossRef][Web of Science][Medline]

20. Saruhan-Direskeneli, G., Esin, S., Baykan-Kurt, B., Ornek I., Vaughan, R. and Eraksoy, M. (1997) HLA-DR and -DQ associations with multiple sclerosis in Turkey. Hum. Immunol., 55, 59–65.[CrossRef][Medline]

21. Ebers, G.C., Kukay, K., Bulman, D.E., Sadovnick, A.D., Rice, G., Anderson, C., Armstrong, H., Cousin, K., Bell, R.B., Hader, W. et al. (1996) A full genome search in multiple sclerosis. Nat. Genet., 13, 472–476.[CrossRef][Web of Science][Medline]

22. Rubio, J.P., Bahlo, M., Butzkueven, H., van der Mei, I.A., Sale, M.M., Dickinson, J.L., Groom, P., Johnson, I.J., Simmons, RD., Tait, B. et al. (2002) Genetic dissection of the human leukocyte antigen region by use of haplotypes of Tasmanians with multiple sclerosis. Am. J. Hum. Genet., 70, 1125–1137.[CrossRef][Web of Science][Medline]

23. Allcock, R.J.N., de la Concha, E.G., Fernandez-Arquero, M., Vigil, P., Conejero, L., Arroyo, R. and Price, P. (1999) Susceptibility to multiple sclerosis mediated by HLA-DRB1 is influenced by a second gene telomeric of the TNF cluster. Hum. Immunol., 60, 1266–1273.[CrossRef][Web of Science][Medline]

24. Fogdell-Hahn, A., Ligers, A., GrØnning, M., Hillert, J. and Olerup, O. (2000) Multiple sclerosis: a modifying influence of HLA class I genes in an HLA class II associated autoimmune disease. Tissue Antigens, 55, 140–148.[CrossRef][Web of Science][Medline]

25. Ebers, G.C. and Paty, D.W. (1980) Studies in familial MS. Trans. Am. Neurol. Assoc., 105, 344–347.[Medline]

26. 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. et al. (2003) HLA-DR2 dose effect on susceptibility to multiple sclerosis and influence on disease course. Am. J. Hum. Genet., 72(3), 710–716.[CrossRef][Web of Science][Medline]

27. Cullen, C.G., Middleton, D., Savage, D.A. and Hawkins, S. (1991) HLA-DR and -DQ DNA genotyping in multiple sclerosis patients in Northern Ireland. Hum. Immunol., 30, 1–6.[CrossRef][Web of Science][Medline]

28. Masterman, T., Ligers A., Olsson T., Andersson, M., Olerup, O. and Hillert, J. (2000) HLA-DR15 is associated with lower age at onset in multiple sclerosis. Ann. Neurol., 48, 211–219.[CrossRef][Web of Science][Medline]

29. Alvarado-de la Barrera, C., Zuniga-Ramos, J., Ruis-Morales, J.A., Estanol, B., Granados J. and Llorente, L. (2000) HLA class II genotypes in Mexican Mestizos with familial and non-familial multiple sclerosis. Neurology, 55, 1897–1900.[Abstract/Free Full Text]

30. Luomala, M., Elovaara, I., Ukkonen, M., Koivula, T. and Lehtimaki, T. (2001) The combination of HLA-DR1 and HLA-DR53 protects against MS. Neurology, 56, 383–385.[Abstract/Free Full Text]

31. Weinshenker, B.G., Santrach, P., Bissonet, A.S., McDonnell, S.K., Schaid, D., Moore S.B. and Rodriguez, M. (1998) Major histocompatability complex class II alleles and the course and outcome of MS: a population-based study. Neurology, 51, 742–747.[Abstract/Free Full Text]

32. Pina, M.A., Ara, J.R., Lasierra, P., Modrego, P.J. and Larrad, L. (1999) Study of HLA as a predisposing factor and its possible influence on the outcome of multiple sclerosis in the sanitary district of Calatayud, Northern Spain. Neuroepidemiology, 18, 203–209.[CrossRef][Web of Science][Medline]

33. Runmarker, B., Martinsson, T., Wahlstrom, J. and Andersen, O. (1994) HLA and prognosis in multiple sclerosis. J. Neurol., 241, 385–390.[CrossRef][Web of Science][Medline]

34. Madigand, M., Oger, J., Fauchet, R., Sabouraud, O. and Genetet, B. (1982) HLA profiles in multiple sclerosis suggest two forms of disease and the existence of protective haplotypes. J. Neurol. Sci., 53, 519–529.[CrossRef][Web of Science][Medline]

35. Siminovitch, K.A. (2005) PTPN22 and autoimmune disease. Nat. Genet., 36, 1248–1249.

36. Begovich, A.B., Caillier, S.J., Alexander, H.C., Penko, J.M., Hauser S.L., Barcellos, L.F. and Oksenberg, J.R. (2004) The R620W polymorphism of the protein tyrosine phosphatase PTPN22 is not associated with multiple sclerosis. Am. J. Hum. Genet., 76, 184–187.

37. Caballero, A., Alves-Leon, S., Papais-Alvarenga, R., Fernandez, O., Navarro, G. and Alonso, A. (1999) DQB1*0602 confers genetic susceptibility to multiple sclerosis in Afro-Brazilians. Tissue Antigens, 54, 524–526.[CrossRef][Web of Science][Medline]

38. Oksenberg, J.R., Barcellos, L.F., Cree, B.A., Baranzini, S.E., Bugawan, T.L., Khan, O., Lincoln, R.R., Swerdlin, A., Mignot, E., Lin, L. et al. (2004) Mapping multiple sclerosis susceptibility to the HLA-DR locus in African Americans. Am. J. Hum. Genet., 74, 160–167.[CrossRef][Web of Science][Medline]

39. Ota, M., Katsuyama, Y., Kimura, A., Tsuchiya, K., Kondo, M., Naruse, T., Mizuki, N., Itoh, K., Sasazuki, T., Inoko H. et al. (2001) A second susceptibility gene for developing rheumatoid arthritis in the human MHC is localized within a 70 kb interval telomeric of the TNF genes in the HLA class III region. Genomics, 71, 263–270.[CrossRef][Web of Science][Medline]

40. Zavattari P., Lampis, R., Motzo, C., Loddo, M., Mulargia, A., Whalen, M., Maioli, M., Angius, E., Todd, J.A. and Cucca, F. (2001) Conditional linkage disequilibrium analysis of a complex disease superlocus, IDDM1 in the HLA region, reveals the presence of independent modifying gene effects influencing the type 1 diabetes risk encoded by the major HLA-DQB1, -DRB1 disease loci. Hum. Mol. Genet., 10, 881–889.[Abstract/Free Full Text]

41. Hudson, T.J. (2003) Wanted: regulatory SNP's. Nat. Genet., 33, 439–440.[CrossRef][Web of Science][Medline]

42. Sadovnick, A.D., Risch N.J., Ebers G.C. and the Canadian Collaborative Study Group. (1998) Canadian collaborative project on genetic susceptibility to MS, phase 2: rationale and method. Can. J. Neurol. Sci., 25, 216–221.[Web of Science][Medline]

43. Olerup, O. and Zetterquist, H. (1992) HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 h: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens, 39, 225–235.[Web of Science][Medline]

44. Spielman, R.S., McGinnis, R.E. and Ewens, W.J. (1993) Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am. J. Hum. Genet., 52, 506–516.[Web of Science][Medline]

45. Risch, N., Spiker, D., Lotspeich, L., Nouri, N., Hinds, D., Hallmayer, J., Kalaydjieva, L., McCague, P. Dimiceli, S., Pitts, T. et al. (1999) A genomic screen of autism: evidence for a multilocus etiology. Am. J. Hum. Genet., 65, 493–507.[CrossRef][Web of Science][Medline]

46. Mantel, N. and Haenszel, W. (1959) Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl Cancer Inst., 22, 719–748.[Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				A. Silva, A Bettencourt, C Pereira, E Santos, C Carvalho, D Mendonca, P. Costa, L Monteiro, and B Martins Protective role of the HLA-A*02 allele in Portuguese patients with multiple sclerosis Multiple Sclerosis, June 1, 2009; 15(6): 771 - 774. [Abstract] [PDF]

				M. R. Lincoln, S. V. Ramagopalan, M. J. Chao, B. M. Herrera, G. C. DeLuca, S.-M. Orton, D. A. Dyment, A D. Sadovnick, and G. C. Ebers Epistasis among HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci determines multiple sclerosis susceptibility PNAS, May 5, 2009; 106(18): 7542 - 7547. [Abstract] [Full Text] [PDF]

				A. Mangalam, D. Luckey, E. Basal, M. Jackson, M. Smart, M. Rodriguez, and C. David HLA-DQ8 (DQB1*0302)-Restricted Th17 Cells Exacerbate Experimental Autoimmune Encephalomyelitis in HLA-DR3-Transgenic Mice J. Immunol., April 15, 2009; 182(8): 5131 - 5139. [Abstract] [Full Text] [PDF]

				S. V. Ramagopalan and G. C. Ebers Epistasis: Multiple sclerosis and the major histocompatibility complex Neurology, February 10, 2009; 72(6): 566 - 567. [Full Text] [PDF]

				M. J. Chao, S. V. Ramagopalan, B. M. Herrera, M. R. Lincoln, D. A. Dyment, A. D. Sadovnick, and G. C. Ebers Epigenetics in multiple sclerosis susceptibility: difference in transgenerational risk localizes to the major histocompatibility complex Hum. Mol. Genet., January 15, 2009; 18(2): 261 - 266. [Abstract] [Full Text] [PDF]

				S. Sawcer The complex genetics of multiple sclerosis: pitfalls and prospects Brain, December 1, 2008; 131(12): 3118 - 3131. [Abstract] [Full Text] [PDF]

				S. V. Ramagopalan, D. A. Dyment, A. D. Sadovnick, and G. C. Ebers HLA-DRB1 AND MULTIPLE SCLEROSIS IN MALTA Neurology, October 21, 2008; 71(17): 1379 - 1379. [Full Text] [PDF]

				M. J. Chao, M. C. N. M. Barnardo, M. R. Lincoln, S. V. Ramagopalan, B. M. Herrera, D. A. Dyment, A. Montpetit, A. D. Sadovnick, J. C. Knight, and G. C. Ebers HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility PNAS, September 2, 2008; 105(35): 13069 - 13074. [Abstract] [Full Text] [PDF]

				S.-M. Orton, A. P Morris, B. M Herrera, S. V Ramagopalan, M. R Lincoln, M. J Chao, R. Vieth, A D. Sadovnick, and G. C Ebers Evidence for genetic regulation of vitamin D status in twins with multiple sclerosis Am. J. Clinical Nutrition, August 1, 2008; 88(2): 441 - 447. [Abstract] [Full Text] [PDF]

				B. Fontaine and L. F. Barcellos Evidence for a complex interaction between HLA-DRB1 and environmental factors in MS Neurology, January 8, 2008; 70(2): 97 - 98. [Full Text] [PDF]

				G. Dean, T. W. Yeo, A. Goris, C. J. Taylor, R. S. Goodman, M. Elian, A. Galea-Debono, A. Aquilina, A. Felice, M. Vella, et al. HLA-DRB1 and multiple sclerosis in Malta Neurology, January 8, 2008; 70(2): 101 - 105. [Abstract] [Full Text] [PDF]

				G. C. DeLuca, S. V. Ramagopalan, B. M. Herrera, D. A. Dyment, M. R. Lincoln, A. Montpetit, M. Pugliatti, M. C. N. Barnardo, N. J. Risch, A. D. Sadovnick, et al. An extremes of outcome strategy provides evidence that multiple sclerosis severity is determined by alleles at the HLA-DRB1 locus PNAS, December 26, 2007; 104(52): 20896 - 20901. [Abstract] [Full Text] [PDF]

				B. M. Herrera, S. V. Ramagopalan, S. Orton, M. J. Chao, I. M. Yee, A. D. Sadovnick, and G. C. Ebers Parental transmission of MS in a population-based Canadian cohort Neurology, September 18, 2007; 69(12): 1208 - 1212. [Abstract] [Full Text] [PDF]

				M. J. Chao, M. C.N.M. Barnardo, G.-z. Lui, M. R. Lincoln, S. V. Ramagopalan, B. M. Herrera, D. A. Dyment, A. D. Sadovnick, and G. C. Ebers Transmission of class I/II multi-locus MHC haplotypes and multiple sclerosis susceptibility: accounting for linkage disequilibrium Hum. Mol. Genet., August 15, 2007; 16(16): 1951 - 1958. [Abstract] [Full Text] [PDF]

				L. F. Barcellos, S. Sawcer, P. P. Ramsay, S. E. Baranzini, G. Thomson, F. Briggs, B. C.A. Cree, A. B. Begovich, P. Villoslada, X. Montalban, et al. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis Hum. Mol. Genet., September 15, 2006; 15(18): 2813 - 2824. [Abstract] [Full Text] [PDF]

				J. A. Traherne, L. F. Barcellos, S. J. Sawcer, A. Compston, P. P. Ramsay, S. L. Hauser, J. R. Oksenberg, and J. Trowsdale Association of the truncating splice site mutation in BTNL2 with multiple sclerosis is secondary to HLA-DRB1*15 Hum. Mol. Genet., January 1, 2006; 15(1): 155 - 161. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 14/14/2019    most recent ddi206v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (56) Request Permissions Google Scholar Articles by Dyment, D. A. Articles by Ebers, G. C. Search for Related Content PubMed PubMed Citation Articles by Dyment, D. A. Articles by Ebers, G. C. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics