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Human Molecular Genetics Advance Access originally published online on June 20, 2007
Human Molecular Genetics 2007 16(16):1951-1958; doi:10.1093/hmg/ddm142
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Transmission of class I/II multi-locus MHC haplotypes and multiple sclerosis susceptibility: accounting for linkage disequilibrium

Michael J. Chao1,2, Martin C.N.M. Barnardo3, Guang-zhi Lui1,2, Matthew R. Lincoln1,2, Sreeram V. Ramagopalan1,2, Blanca M. Herrera1,2, David A. Dyment1,2, A. Dessa Sadovnick4 and George C. Ebers1,2,*

1 Department of Clinical Neurology, University of Oxford, Level 3, West Wing, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK, 2 Welcomes Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK, 3 Oxford Transplant Centre, Nuffield Department of Surgery, Churchill Hospital, Oxford OX3 7LJ, UK and 4 Division of Neurology, Department of Medical Genetics and Faculty of Medicine, University of British Columbia, Vancouver, Canada V6T 1Z4

* To whom correspondence should be addressed. Tel: +44 1865231903; Fax: +44 1865231914; Email: george.ebers{at}clneuro.ox.ac.uk

Received December 19, 2006; Revised April 10, 2007; Accepted May 31, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 REFERENCES
 
The human major histocompatibility complex (MHC) class II region is associated with genetic susceptibility to multiple sclerosis (MS). Roles for HLA class I loci have been supported in several case–control studies, but this methodology does not consider the known linkage disequilibrium (LD) between class I and II loci. In 1258 individuals from 294 MS families, we analysed class I and II interactions. Using transmission disequilibrium test and haplotype analyses, we found positive associations between MS and several HLA-DRB1*15-HLA-A haplotypes including HLA-DRB1*15-HLA-A*02 (P = 2.41 x 10–5) and -HLA-A*03 (P = 8.42 x 10–6) and several HLA-DRB1*15-HLA-B haplotypes including HLA-DRB1*15-HLA-B*07 (P = 2.23 x 10–10). HLA-DRB1*15 haplotypes divergent for reported HLA-A allelic associations were equally over-transmitted, illustrating no detectable effect of HLA-A or -B alleles in cis on susceptibility. HLA-A and -B alleles on haplotypes not bearing HLA-DRB1*15 were not over-transmitted. Similarly, general over-transmission of HLA-DRB1*15 haplotypes was independent of the HLA-B allele present. Furthermore, HLA-B*07 haplotypes from HLA-DRB1*X-HLA-B*X/HLA-DRB1*X-HLA-B*07 heterozygous parents were transmitted per random expectation giving no indication of HLA-B independence or trans complementation of HLA-DRB1*15 by HLA-DRB1*X-HLA-B*07 haplotypes. These results imply that many reports of class I allelic associations in MS are class II dependent, secondary to LD with class II loci. The lack of independent class I associations suggests that virus-related class I-antigen complexes are not T-cell targets in MS. The inability to replicate confirmed case–control associations highlights the importance of family-based analyses. The frequency of allelic associations not being replicated emphasizes the requirement for constructing multi-locus haplotypes in dissecting associations in regions of tight LD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 REFERENCES
 
Multiple sclerosis is a complex trait in which autoimmunity results in nervous system injury and axonal loss. The precise aetiology of MS is not yet well understood, however, both genetic and environmental factors are implicated (1–6). Apart from the human MHC genomic region (6p21.3), no other loci for susceptibility have been unambiguously confirmed (7–10). Loci of very small effect which would not influence the inheritance pattern have not been excluded. The MHC is a difficult region to dissect due to its strong but highly variable patterns of LD. Initial associations with MS were observed with the human leukocyte antigen (HLA) class I alleles HLA-A*03 and HLA-B*07 of the MHC (11,12). These were subsequently reported to be secondary to suggested primary associations with the HLA class II extended haplotype HLA-DRB5*0101-HLA-DRB1*1501-HLA-DQA1*0102-HLA-DQB1*0602 (13,14). The association between MS and the HLA class II HLA-DRB1*1501 haplotype is now well established (5–7,15). However, the mechanism of contribution remains unclear as does the proportion of the genetic contribution attributable to this locus, the role of the HLA class I region and the way in which these two loci might interact.

Large numbers were needed to demonstrate allelic heterogeneity at HLA-DRB1 and striking interactions between different HLA-DRB1 alleles (5). These included the susceptibility alleles (HLA-DRB1*15 and HLA-DRB1*17), resistance alleles (HLA-DRB1*01, HLA-DRB1*11 and HLA-DRB1*14) and class II haplotype interactions in trans. More specifically, HLA-DRB1*01 and HLA-DRB1*08 alleles were found to interact with HLA-DRB1*15, and HLA-DRB1*01 was significantly under-transmitted to offspring in the presence of HLA-DRB1*15, whereas HLA-DRB1*08 was transmitted preferentially with HLA-DRB1*15 (5). These developments have made direct examination of class I necessary.

Increased HLA haplotype sharing has been reported in a large data set of MS families where parents lacked the HLA-DRB1*15 allele (16). This has implicated involvement of another tightly linked locus or even adjacent regulatory regions.

As well as the established HLA class II associations, accumulating evidence has suggested that HLA class I loci may also influence susceptibility. By using microsatellite markers in the MHC (17,18), one study of Sardinian MS families identified a region telomeric of HLA class I showing association independent of HLA class II (17). Another study consisted of Tasmanian MS cases and controls identified a similar HLA class I region that independently increased risk of and provided protection from MS (18).

Three additional reports suggested a primary role for HLA class I independent of class II (19–21). The class I HLA-A*03 allele was found to increase MS risk, while HLA-A*02 and HLA-C*05 alleles had protective effects independent of HLA-DRB1*15 (19,21). However, allele frequencies of MS cases and controls lacked parental information, an approach potentially less informative than family-based association analyses. Further evidence suggested that HLA class I alleles modulate class-II associated risk (20) and proposed differences between HLA-A and HLA-B. In both instances, association with HLA-B*07 was believed to be secondary to the HLA class II association (19,20), but not for HLA-A alleles.

Using logistic regression analysis of single nucleotide polymorphisms (SNPs) spanning the MHC, a more recent study has shown that HLA class II was solely responsible for the MHC association, and involvement of loci outside this region was not supported by this data; however, class I loci themselves were not typed in this study. An important lesson from this SNP study (6) was that a highly polymorphic locus such as HLA class II could be more informative than even extended SNP haplotypes. For loci with many functional alleles, direct typing could detect effects not apparent with the SNP panel used despite its density. Therefore, the present study aimed to comprehensively analyse the role of HLA class I and class II regions in MS susceptibility by directly typing selected HLA class I loci in a large sample of multiplex families already typed for class II.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 REFERENCES
 
Transmissions of HLA-DRB1*15 and other HLA-DRB1 alleles in the HLA-A and HLA-B typed families
In the subset of families with HLA-DRB1*15-positive parents typed here for class I alleles (vide infra), we show in Table 1 for baseline purposes that affected offspring showed positive association with the HLA-DRB1*15 allele, with this allele being transmitted 284 times and not transmitted 116 times (P = 1.44 x 10–17). HLA-DRB1*01 was under-transmitted in families with HLA-DRB1*15 positive and negative parents. In addition to the under-transmission of HLA-DRB1*01, several other HLA-DRB1 alleles were significantly under-transmitted, which included HLA-DRB1*07, *09, *11, *12, *14 and *16 (Table 1). Total HLA-DRB1*X transmissions were 355 times transmitted and 518 times not transmitted (P = 3.47 x 10–8). There was no significant transmission distortion observed for the totalled HLA-DRB1*X allele in HLA-DRB1*15-negative families (Table 1).


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  		Table 1. Transmission data of HLA-DRB1 alleles in families with HLA-DRB1*15 positive parents and families with HLA-DRB1*15 negative parents

 
Transmissions of HLA-A*02, HLA-A*03, HLA-B*07 and other HLA-A and HLA-B alleles
There were no significant transmission distortions observed for HLA-A*02 in families with or without HLA-DRB1*15-positive parents (Table 2). The HLA-A*03 allele was significantly over-transmitted in families with HLA-DRB1*15-positive parents (transmitted 107 times and not transmitted 72 times; P = 0.0087) (Table 2). HLA-A*33 appeared to be significantly under-transmitted in HLA-DRB1*15-positive families. HLA-A*11 seemed to be over-transmitted, whereas HLA-A*23 and HLA-A*30 were under-transmitted in HLA-DRB1*15-negative families (Table 2). For the totalled HLA-A*X allele (where *X was any allele other than *02 or *03), significant transmission distortion was observed in neither HLA-DRB1*15 positive nor negative families (Table 2).


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  		Table 2. Transmission data of HLA-A alleles in families with HLA-DRB1*15-positive parents and families with HLA-DRB1*15-negative parents

 
The HLA-B*07 allele was significantly over-transmitted in families with HLA-DRB1*15-positive parents, (transmitted 174 times and not transmitted 95 times; P = 1.22 x 10–06) (Table 3). There were relatively few HLA-B*07-positive HLA-DRB1*15-negative families because of strong HLA-DRB1-HLA-B LD (Table 3). Several HLA-B alleles were under-transmitted (P-values uncorrected for multiple testing), which included HLA-B*38, B*52, B*55 and B*60 (Table 3). The total HLA-B*X results were 398 alleles transmitted and 471 not transmitted (P = 0.013), reflecting the over-transmission of HLA-B*07. There was no significant transmission distortion observed for the totalled HLA-B*X allele in HLA-DRB1*15 negative families (Table 3).


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  		Table 3. Transmission data of HLA-B alleles in families with HLA-DRB1*15-positive parents and families with HLA-DRB1*15-negative parents

 
HLA-DRB1*15-HLA-A and HLA-DRB1*X-HLA-A haplotype interactions
All HLA-DRB1*15-HLA-A haplotypes appeared to be over-transmitted from parents to affected offspring, and a number of haplotypes were significantly over-transmitted, including HLA-DRB1*15-HLA-A*01 (15-1: TR = 27, NT = 14, P = 0.041), -A*02 (15-2: TR = 74, NT = 31, P = 2.41 x 10–5), -A*03 (15-3: TR = 78, NT = 32, P = 8.42 x 10–6), -A*24 (15-24: TR = 45, NT = 13, P = 1.54 x 10–5), -A*25 (15-25: TR = 10, NT = 3, P = 0.046) haplotypes (Table 4). This was not the case in the presence of other HLA-DRB1 alleles in cis, except for the totalled HLA-DRB1*X-HLA-A*11 haplotype, which appeared to be over-transmitted in HLA-DRB1*15-negative families. The HLA-DRB1*X-HLA-A*02 haplotype was significantly under-transmitted in HLA-DRB1*15-positive families (X-2: TR = 84, NT = 135, P = 0.00060), but this did not differ significantly from the totalled HLA-DRB1*X-HLA-A haplotypes excluding HLA-DRB1*X-HLA-A*02 ({chi}2 = 1.26; NS) and there was no significant transmission distortion observed for this haplotype in HLA-DRB1*15-negative families (X-2: TR = 34, NT = 29, P = 0.53) (Table 4).


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  		Table 4. TDT comparison between HLA-DRB1*15-HLA-A haplotypes and HLA-DRB1*X-HLA-A haplotypes

 
In HLA-DRB1*15-positive families, the total transmission of HLA-DRB1*15-HLA-A haplotypes was 255 times transmitted and 104 times not transmitted (P = 1.60 x 10–15), whereas the total transmission of HLA-DRB1*X–HLA-A haplotypes was 256 times transmitted and 363 times not transmitted (P = 1.70 x 10–5). There was no significant transmission distortion observed for the totalled HLA-DRB1*X–HLA-A haplotype in HLA-DRB1*15-negative families (Table 4).

HLA-DRB1*15-HLA-B and HLA-DRB1*X-HLA-b haplotype interactions
Parallel to the transmission results of HLA-DRB1*15-HLA-A haplotypes shown above, HLA-DRB1*15-HLA-B haplotypes appeared to be generally over-transmitted from parents to affected offspring (Table 5). Several were significantly over-transmitted, including HLA-DRB1*15-HLA-B*07 (15-7: TR = 149, NT = 59, P = 2.23 x 10–10), -B*18 (15-18: TR = 16, NT = 4, P = 0.0055), -B*40 (15-40: TR = 12, NT = 4, P = 0.041), -B*51 (15-51: TR = 12, NT = 2, P = 0.0049), and -B*57 (15-57: TR = 11, NT = 2, P = 0.0088) haplotypes. HLA-B alleles were over-transmitted only in HLA-DRB1*15-bearing haplotypes, and not with other HLA-DRB1 alleles (Table 5).


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  		Table 5. TDT comparison between HLA-DRB1*15-HLA-B haplotypes and HLA-DRB1*X-HLA-B haplotypes

 
In HLA-DRB1*15-positive families in total, HLA-DRB1*15-HLA-B haplotypes were 259 times transmitted and 112 times not transmitted (P = 2.31 x 10–14), whereas HLA-DRB1*X-HLA-B haplotypes were 214 times transmitted and 304 times not transmitted (P = 7.66 x 10–5). There were no significant transmission distortions observed for the totalled HLA-DRB1*X-HLA-B haplotypes in HLA-DRB1* 15-negative families (Table 5).

Finally, we examined transmissions of HLA-B*07 from HLA-DRB1*X-HLA-B*X/HLA-DRB1*X-HLA-B*07 heterozygous parents (where *X is any allele other than HLA-DRB1*15 and HLA-B*07). Among 49 such instances, we found no transmission distortion for the HLA-DRB1* X-HLA-B*07 haplotypes (TR = 25, NT = 24).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 REFERENCES
 
The evidence for genetic influence on MS susceptibility is very strong; however, it has not been possible to unambiguously identify loci outside the well-known MHC region (9). Extensive genome searches have been negative in firmly establishing other loci, and, recently, the paradigm of susceptibility being determined by many loci of small effect supported by some research groups (10,22) has been called into question (9). Accordingly, the one unambiguous region of susceptibility in the MHC warranted more scrutiny to determine if complexity implied by the genetic epidemiology is contained within the MHC itself.

Independent HLA class I associations have been suggested by several case–control studies (19–21). Using allele frequencies of cases and controls, a Swedish study (19) demonstrated that the presence of HLA-A*02 decreased the risk and the HLA-A*03 increased the risk of MS, independently of HLA-DRB1*15.

In addition, by LD mapping, a Sardinian microsatellite-based study identified a region telomeric of the class I region demonstrating association independent of class II (17). Parallel findings were reported by an Australian case–control study, which used microsatellite markers to study the class I region believed to play an independent role in susceptibility to and protection from MS (18).

To help resolve the question of whether HLA class I exerts an independent effect, it has been necessary to use family-based transmission data and to derive two- and three- locus haplotypes. The well-documented HLA-DRB1*15 association, supported by previous studies (7–10,15) was accompanied by significant under-transmissions of HLA-DRB1*01, *07, *09, *11, *12, *14 and *16 in HLA-DRB1*15-positive families in the class-I typed subset studied here as described (15). Previous observations of interactions between HLA-DRB1 alleles have been extended to evaluate interactions between HLA-DRB1, HLA-A and HLA-B haplotypes.

As expected, we found positive associations with HLA-DRB1*15-HLA-A and HLA-DRB1*15-HLA-B haplotypes. However, haplotype transmission disequilibrium test (TDT) analyses illustrated that HLA-DRB1*15 haplotypes are over-transmitted regardless of what common allele is present at HLA-A and -B. Comparisons of HLA-A*02, HLA-A*03 and HLA-B*07 allele frequencies on HLA-DRB1*15 and HLA-DRB1*X haplotypes themselves reflect the LD between HLA class II and class I. For example, HLA- DRB1*15-HLA-A*03 is one of the most frequent haplotypes transmitted in families with HLA-DRB1*15-positive parents, but HLA-DRB1*X-HLA-A*03 haplotypes are much less frequent in families with HLA-DRB1*15-negative parents, implying these HLA-A findings might be a secondary effect.

The uniform over-transmission of HLA-DRB1*15-HLA-A*X alleles (where X is any common A* allele and encompassing HLA-DRB1*X-HLA-A*02 and HLA-DRB1*X-HLA-A*03 haplotypes) in HLA-DRB1*15-positive families contrasts with results in the HLA-DRB1*15 negatives. Here transmissions of HLA-A*02 and HLA-A*03 were not altered from chance expectation as they would have been if they had exerted an effect independent of HLA-DRB1*15. The HLA-DRB1* 15-HLA-A*02 and HLA-DRB1*15-HLA-A*03 haplotypes were similarly and significantly over-transmitted from parents to affected offspring (Table 4). Therefore the presence on HLA-DRB1*15 haplotypes of HLA-A alleles at polar extremes of allelic association in the Swedish case–control material did not differentiate among haplotype transmissions in our data. HLA-A*02 was perhaps under-transmitted to a greater degree than A*03 or any other allele on HLA-DRB1*X bearing haplotypes but transmission was random when the effect of HLA-DRB1*15 was removed by enumerating transmissions in HLA-DRB1*15-negative families (Table 4). Furthermore, the overall under-transmission of HLA-DRB1*X two-locus haplotypes (secondary to the effect of HLA-DRB1*15 over-transmission from heterozygous parents) was nearly identical for HLA-A (OR = 0.71) and HLA-B (OR = 0.70). These findings are also against any independent effect of the HLA-A locus, but we cannot rule out an interactive HLA-DRB1*15-dependent effect of the HLA-A*02 allele in trans. However, since there is already clear evidence for interactions at class II which can suppress the effect of HLA-DRB1 alleles on susceptibility (5), this calls for additional analysis controlling for the presence of specific HLA-DRB1 alleles on HLA-A*02-bearing haplotypes.

These results show how allele frequencies can be potentially misleading for association in the absence of confirmatory transmission analysis possible by having available parents. Therefore, this Canadian cohort could confirm neither the independent protective effect of HLA-A*02 nor the independent association of HLA-A*03 that have been previously reported (19,20).

The HLA-A*11 allele appeared to be over-transmitted in families negative for HLA-DRB1*15 (Tables 2 and 4), although significance would not remain after correction. Additionally, no transmission distortion of HLA-A*11 (or other class I alleles) in the absence of HLA-DRB1*15 was observed in an expanded cohort of more than 3000 individuals typed for HLA class I (unpublished data).

Results from the study of HLA-A alleles implied that reported associations of the HLA-B*07 allele (19,20) could also be secondary. However, the HLA-B*07 allele may differ from HLA-A*02 and HLA-A*03 at least insofar as there is much stronger LD between the HLA-B locus and HLA-DRB1*15. Furthermore, transmission of the HLA-DRB1* 15-HLA-B*07 haplotypes was slightly greater than for non-B*07 HLA-DRB1*15 haplotypes (OR = 2.53 versus OR = 2.10, Table 5). However, the presence of general over-transmission of HLA-DRB1*15 haplotypes, largely irrespective of the allele present at HLA-B, would imply that any other HLA-B associations could also be secondary to the primary association at DR or DQ. Transmissions in the HLA-DRB1*15-positive families cohere with this since HLA-DRB1*X-HLA-B*07 haplotypes are under-transmitted (OR = 0.76) as for other HLA-DRB1*X-HLA-B haplotypes (OR = 0.70) as shown in Table 5 while this should be expectedly less apparent or even over-transmitted if there were an independent effect in trans of this HLA-B allele.

In contrast to HLA-A, we could not adequately address transmission of HLA-B*07 alleles in HLA-DRB1*15-negative families since there were many fewer transmissions observable. There are few HLA-B*07-positive haplotypes among the HLA-DRB1*15-negative parent group. Therefore, we examined transmission of HLA-B*07 haplotypes from the 49 parents with MS offspring who had HLA-B*07 but not HLA-DRB1*15 on either of their haplotypes, thereby removing the confounding influence of HLA-DRB1*15. In this analysis which allows a more parallel comparison to the other HLA-A alleles, HLA-DRB1*X-HLA-B*07 haplotypes were randomly transmitted from HLA-DRB1*X-HLA-B*07/HLA- DRB1*X-HLA-B*X parents (where *X is any allele other than HLA-DRB1*15 and HLA-B*07). Similarly, diminished transmission of HLA-DRB1*X-HLA-B*07 from HLA-DRB1* X-HLA-B*07/HLA-DRB1*15-HLA-B*X parents did not differ from that seen for other HLA-B alleles. Therefore, we could detect no trans complementation of HLA-B*07 with HLA-DRB1*15 or with HLA-DRB1*X haplotypes.

We did not study alleles at the HLA-C locus but since it is very closely linked to HLA-B, distorted case–control frequencies at this locus would need careful exclusion of LD as an explanation (21,22). We have not included DQ data here but the very tight LD between DR and DQ implies little additional information would be added on the question of HLA class I independence.

There were minor differences among the HLA-DRB1* 15-HLA-A and –HLA-B haplotypes and some indication that high and low frequency HLA-A and -B alleles associated inversely with transmission. However, the number of transmissions of rare alleles was too small to make certain and this post-hoc observation would require independent replication under this specific a priori hypothesis.

It is possible that there could be differences among populations with respect to the role of class I, however, it may take additional family-based studies in Swedish, Australian and Sardinian patients to prove this. In the meantime, the case–control results cannot be entirely dismissed. When comparing the Canadian and Swedish populations, we note that the Canadian patients have identical HLA-A*02 frequencies when compared with the Swedish MS patients. However, the Swedish normal controls had an over-represented HLA-A*02 frequency when compared with the Canadian normal controls. The differences in controls appear to be responsible for the reported finding of an independent protective effect of HLA-A*02 in the Swedish case–control cohort. The control differences between the Swedish and the much more heterogeneous Canadian population identifies an interesting divergence of common class I allele frequencies of uncertain origin in the two populations.

Because of the family-based nature of the haplotype TDT, our results clarify the relationship between HLA class I and class II at least for the Canadian population, largely Northern European derived. Our results do not rule out a class II-dependent functional interaction for class I, but imply that the HLA class II region is responsible for secondary HLA class I allelic associations arising from LD. The class I haplotypes may, however, shed some light on heterogeneity among class II alleles and their associated haplotypes in MS susceptibility and on the more general vexing conundrum of the long range discontinuous LD encompassing class I and class II loci in this region.

The exceptionally and unprecedentedly high frequency of non-reproducibility of association results (23) has drawn increased attention from general scientific commentators (24) and the results reported here suggest that type I errors are not necessarily excluded by replication of specific gene association, if this remains secondary to linkage disequilibrium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
NOTE ADDED IN PROOF
 REFERENCES
 
Subjects
We selected 294 multiplex MS families (Individuals; n = 1258) as part of an ongoing Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS), for which the methodology has been described previously (25). All families were Canadian and of European descent.

HLA typing
The genotyping for the HLA-DRB1 was performed using either low- or high-resolution allele-specific polymerase chain reaction (PCR) amplification method (5). Low-resolution HLA-DRB1 genotypes were obtained by a combination of 24 PCR reactions, and high-resolution HLA-DRB1 genotypes were obtained with an additional 48 PCR reactions. In each individual reaction, positive control primers were designed to amplify a second non-polymorphic genomic control segment. Amplified products were separated by electrophoresis in 2% agarose gels containing ethidium bromide after the addition of loading buffer, and visualized them using ultraviolet (UV) illumination.

Only a small minority of HLA-DRB1 typing was performed using low-resolution allele-specific PCR amplification method after establishing in several thousand individuals with high resolution, and in this paper, the allele HLA-DRB1*15 refers to the subtype HLA-DRB1*1501, which is the most common HLA-DRB1*15 found in Caucasians.

The genotyping for the HLA-A and HLA-B were performed using a low-resolution allele-specific PCR amplification method (26). Low-resolution HLA-A and HLA-B genotypes were obtained by a combination of 96 PCR reactions. PCR products were electrophoresed in 1% agarose gels containing ethidium bromide after the addition of loading buffer and visualized using UV illumination.

Statistical methods
The family pedigree files were first tested using the PEDCHECK program (27) for the presence of errors in Mendelian transmission. TDT was performed for each locus individually and also to multi-locus haplotypes using the TDTPHASE program of the UNPHASED software package (28, http://www.hgmp.mrc.ac.uk/). In the two-locus haplotype transmission analyses (HLA-DRB1-HLA-A and HLA-DRB1-HLA-B haplotypes), we have not listed HLA-DRB1*15-bearing haplotypes (and their paired HLA-DRB1*X haplotypes) with less than 10 transmissions. Fisher's exact test was used when the expected transmission in any of the cells of the table was below five. Confidence intervals (CI, 95%) of odds ratios (OR) were calculated and are presented in all tables of results. We did not calculate 95% CIs for the rare alleles/haplotypes. Families had been selected for those with parents typed but in a minority, parents who were not genotyped were reconstructed whenever possible from unaffected siblings only. In cases when one parent was unknown, only those instances where both the genotyped parent and the affected offspring were heterozygous for different alleles were the transmissions counted in order to avoid directional bias.

Apart from the well-established HLA-DRB1*15 association, we did not find additional susceptibility at the HLA class I region. Since this study reports negative results of HLA class I association, correction for multiple testing was not applied, and all P-values in tables were presented as uncorrected P-values (Puncorrected).


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
REFERENCES
 
MS association with SNPs in the interleukin-7 receptor (ILT-R) has been confirmed (J. Hillert-personal communication).


   ACKNOWLEDGEMENTS
 
We would like to thank our colleagues at the Wellcome Trust Centre for Human Genetics and also the Oxford Transplant Centre for their help and support. Informed consent was obtained from all subjects and the experiments performed for this investigation comply with current guidelines and ethics. This study was made possible by the Canadian Collaborative Project on the Genetic Susceptibility to MS (CCPGSMS), and was funded by the MS Society of Canada.

Canadian Collaborative Project Group members: Vancouver: J. J.-F. Oger, D.W. Paty, S.A. Hashimoto, V. Devonshire, J. Hooge, J.P.Smythe and T. Traboulsee; Calgary: L. Metz; Edmonton: S. Warren; Saskatoon: W. Hader; Ottawa: R. Nelson and M. Freedman; Kingston: D. Brunet; Hamilton: J. Paulseth; London: G. Rice and M. Kremenchutzky; Toronto: P. O'Connor, T. Gray and M. Hohol; Montreal: P. Duquette and Y. Lapierre; Quebec City: J.-P. Bouchard; Halifax: T.J. Murray, V. Bhan and C. Maxner; St Johns: W. Pryse-Phillips and M. Stefanelli.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 REFERENCES
 

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

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

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