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Human Molecular Genetics, 2004, Vol. 13, No. 10 1005-1015
DOI: 10.1093/hmg/ddh123
Human Molecular Genetics, Vol. 13, No. 10 © Oxford University Press 2004; all rights reserved

An extended genome scan in 442 Canadian multiple sclerosis-affected sibships: a report from the Canadian Collaborative Study Group{dagger}

David A. Dyment1, A. Dessa Sadovnick2, Cristen J. Willer1, Holly Armstrong3, Zameel M. Cader1, Steven Wiltshire1, Bernadette Kalman4, Neil Risch5 and George C. Ebers1,*

1The Wellcome Trust Center for Human Genetics, Oxford OX37BN, UK, 2Department of Medical Genetics and Faculty of Medicine (Division of Neurology), University of British Columbia, Vancouver, Canada V6T 2B5, 3Department of Clinical Neurology, University of Western Ontario, London, Canada N6A 5A5, 4Department of Neurology, Columbia University, New York, NY 10019, USA and 5Department of Genetics, Stanford University, Palo Alto, CA 94305-5120, USA

Received December 5, 2003; Revised February 19, 2004; Accepted March 23, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple sclerosis (MS) is a complex trait with a sibling relative risk ({lambda}sibs) between 18 and 36. We report a multistage genome scan of 552 sibling pairs from 442 families, the largest MS family sample assessed for linkage. The first stage consisted of a genome scan for linkage with 498 microsatellite markers at an average spacing of 7 cM in 219 sibling pairs. The second stage involved further genotyping of markers from positive regions in an independent sample of 333 affected sibling pairs. The global distribution of allele sharing for all markers showed a shift towards greater sharing within the affected sibling pair group but not in the discordant sibling pair group. This shift indicates that the number of contributing genetic factors is likely to be moderate to large. Only markers at chromosome 6p showed significant evidence for linkage (MLOD=4.40), while other regions were only suggestive (1p, 2q, 5p, 9q, 11p, 12q, 18p, 18q and 21q) with MLODs greater than 1.0. The replication analysis involving all 552 affected sibling pairs confirmed suggestive evidence for five locations, namely, 2q27 (MLOD=2.27), 5p15 (MLOD=2.09), 18p11 (MLOD=1.68), 9q21 (MLOD=1.58) and 1p31 (MLOD=1.33). Suggestive linkage evidence for a previously reported location on chromosome 17q (MLOD=1.67) and a prior association with marker D17S789 was replicated. We showed that the overall excess allele sharing we observed for the entire sample was due to increased allele sharing within the DRB1*15 negative subgroup alone. This observation is most consistent with a model of genetic heterogeneity between HLA and other genetic loci. These findings offer guidance for future genetic studies including dense SNP linkage disequilibrium analysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). Demyelination and nerve fiber degeneration are cardinal features of this disease (1). Focal damage to the myelin and selective axon loss appear to be responsible for the varied clinical symptoms observed in MS, including fatigue, visual impairment, weakness, sensory disturbances and impaired cognitive function among others (1).

The cause of MS remains unknown, although environmental, genetic and stochastic factors are involved. In Canada, ~20% of MS patients have at least one first, second or third degree relative with MS (2). Given the Canadian lifetime population risk of ~0.1–0.2%, the population relative risk for the siblings of MS patients (or {lambda}sibs) can be estimated at 18–36. The involvement of genes in MS susceptibility is shown by the marked difference in the monozygotic twin rate compared with the dizygotic twin rate (3–6). The extent of this genetic contribution is clearly indicated by the results of adoptee (7) and half-sibling studies (8). Together, these studies show that familial aggregation of MS is due primarily to genetic sharing between relatives rather than the shared familial microenvironment.

Candidate genes have been chosen for study because of their involvement in various immune/inflammatory processes. With the exception of the major histocompatibility complex (MHC) and perhaps the T-cell receptor ß locus, this approach has not yet had much success. The MS-associated MHC haplotype is the HLA class II DQA1*0102–DQB1*0602–DRB1*1501–DRB5*0101 (9). However, if haplotype sharing is a valid measure of the magnitude of effect, this region can account for only 14% of risk (10).

Several genome scans in multiplex MS families have been performed to identify other susceptibility genes (11–18). A number of non-MHC regions potentially containing susceptibility genes have been identified, including 17q22–24, 6q27, 12q23–24 and 19q13. However, no novel susceptibility genes have yet been discovered (19,20). These studies may have been underpowered to detect mild to moderate susceptibility loci (21) and/or genetic heterogeneity among sibling pair families has confounded these analyses.

The study presented here is the largest to date and extends genome search data with more genomic markers and sibling pair families.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The genome-wide multipoint scores and the exclusion plots for a locus contributing a lambda value ({lambda}locus)>=2.0 are presented in Figure 1. Seventy-seven percent of the genome could be excluded (LOD<–2) as containing a locus with {lambda}>=2.0 and 96% could be excluded at {lambda}>=3.0.




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  		Figure 1. Linkage results in sibling pairs: red line, MLODS in 219 sibling pairs; blue line, MLODS in 552 sibling pairs; black line, exclusion at {lambda}>=2.0.

 
Nine regions had a multipoint MLOD score of 1.0 or greater in datasets 1–3 (Table 1). These regions are on chromosomes 1p31–32, 2q37–qtel, 5p15–ptel, 6p21, 9q21, 11p13–15, 12q21–23, 18p11 and 18q23 (Table 2). Chromosome 21q22 had an MLOD of 0.98 and was also included for further genotyping and analysis. No marker achieved an MLOD of 3.37 corresponding to an empirical genome-wide significance value of 0.05. The highest MLOD was 2.25 for HLA DRB1 of the MHC on chromosome 6p21. These results are available at http://www.well.ox.ac.uk/ebers/.


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  		Table 1. Characteristics of family material
 

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  		Table 2. The highest scores (MLOD>1.0) in genome-wide scan of 219 sibling pairs and the results of a follow-up in 333 sibling pairs (total=552)
 
For a robust examination of our linkage results, we compared allele sharing for concordant (affected–affected) versus discordant (affected–unaffected) sibling pairs as described previously (22). There were 77 discordant sibling pairs (DSPs) counted in the initial 172 families. A DSP was counted using the first unaffected sibling in the sibship and only one discordant pair was counted per family. The amount of allele sharing at 481 markers spanning the autosomes in the DSPs was compared to the affected sibling pairs (ASPs) (datasets 1–3; n=219). For the ASPs there was a total of 49 138/96 752 alleles shared in common (50.8%). The 77 DSPs had a total of 9102/18 254 alleles shared (49.9%). Following this, Z-scores were calculated separately for each marker for both the ASPs and DSPs and then ranked. Figure 2 plots the ranked Z-scores for both groupings. Of the 481 markers, 11 had Z-scores>1.96 in the DSPs. This is as expected by chance (0.025x481=12.025). For the ASPs there were 22 markers with Z-scores>1.96; greater than expected by chance. This result supports genetic susceptibility to MS likely involving multiple loci. The greatest score was Z=3.37 for the DRB1 locus.



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  		Figure 2. Global distribution of Z-scores in ASPs and DSPs: red line, DSPs Z-scores; blue line, expected Z-scores; black line, ASPs Z-scores.

 
Follow-up: DS4 and 5 and the combined sample
The 10 regions identified in the first stage analysis (MLOD>=1.0) were further genotyped in a second sample of 333 sibling pairs (datasets 4 and 5). A total of 35 markers were genotyped at this point; the 10 markers with MLOD>=1.0 plus the additional, flanking markers used in the initial scan. The markers with the highest score per region are presented in Table 2.

Chromosome 6 follow-up
Of the original 10 regions followed-up, only HLA DRB1 had an MLOD score over 3.0 (MLOD=3.10) in the follow-up datasets. In the combined datasets 1–5, the multipoint MLOD for HLA DRB1 was 5.30. This differs from the MLOD of 4.09 reported previously for HLA DRB1 (10) as here it was the result of a multipoint analysis incorporating adjacent microsatellite markers (D6S273, D6S276, D6S461). An additional 10 microsatellites located at 6p21 were genotyped in the combined 552 sibling pairs. Upon re-analysis with the combined 13 microsatellites and HLA DRB1, the highest MLOD was still at HLA DRB1 but decreased from 5.30 to 4.40 (Fig. 1). The MLOD scores for MHC peaked at the HLA DRB1 locus but a second peak was observed telomeric to the HLA Class I region (Fig. 1). The MLOD for D6S285 was 3.66 with 59% sharing and is 6 cM telomeric from D6S461, a marker originally shown to have positive evidence for linkage and association in our initial genome scan (11). The MLOD for D6S461 is 3.30 and has 57.5% sharing.

Chromosome 2 follow-up
The next highest MLOD observed in the follow-up sample was for marker D2S2317 at chromosome 2q which is 30 cM from marker D2S140 highlighted in stage 1. D2S2317 showed 57.6 % sharing and an MLOD of 1.60 in datasets 4 and 5, but only 52% (MLOD=0.11) sharing in datasets 1–3. In the total sample of 552 sibling pairs, the multipoint MLOD for D2S2317 decreased to 1.27. However, the evidence for linkage at adjacent markers telomeric to D2S2317 increased; the MLODs for D2S338, D2S2253 and the original marker D2S140 were 1.86, 1.96 and 2.11, respectively (Table 2). Three additional markers, D2S125, D2S395 and D2S2338 were then genotyped in datasets 1–5 and the multipoint MLOD for 2q27 increased to 2.27 for D2S2338 (Fig. 1). D2S2338 is 5.5 cM from the peak observed in the first stage scan at D2S140.

Chromosome 5 follow-up
Three microsatellite markers (D5S405, D5S406, D5S635) located at 5p have been previously genotyped and analyzed in a follow-up linkage study (23). The same three markers were typed here in dataset 5 resulting in a mild positive MLOD for D5S405 (MLOD=0.30). For the combined families and additional markers spanning 5p, the highest MLOD was observed for D5S2005 at 5p15 (Table 2); the MLOD was 1.89 with 57.5% sharing. At this stage an additional nine microsatellites located within 5p15 were genotyped in all families and the MLOD for D5S2005 increased from 1.89 to 2.09 (Fig. 1).

Chromosome 1 and 9 follow-up
The markers on chromosomes 1p31–32 and 9q21 were also positive in datasets 4 and 5. The MLOD for marker D1S230 was 0.35 in these two datasets and increased to an MLOD of 1.33 in the combined sample. D9S301 at 9q21 had an MLOD of 0.37 for datasets 4 and 5 and increased from 1.26 to 1.58 in the total families (Table 2). However, the remaining five regions of the original 10 highlighted regions with MLOD over 1.0 (11p13–15, 12q21–23, 18p11, 18q23 and 21q22) showed little or no evidence of linkage in the follow-up families (Table 2). The results of an identity-by-state analysis were comparable using the sib_phase program of ASPEX.

Chromosome 17 follow-up
Another eight microsatellite markers on chromosome 17q were genotyped in datasets 4 and 5. Markers in the region did not initially attain the arbitrary cut-off of MLOD>=1.0 in datasets 1–3. The highest multipoint score was 0.76 with 55.8% sharing at marker D17S785. However, based on suggestive linkage results from UK and Finnish scans (12,14), prior Canadian studies (23) and subsequent significant linkage to the region (19), further genotyping in Canadian families was warranted. For datasets 4 and 5 the highest MLOD was 0.89, again for D17S785. For the total 552 sibling pairs the MLOD was 1.67 with 56.0% sharing observed.

In summary, six regions of the original 11 regions that were followed-up in datasets 4 and 5 showed increased evidence for linkage, while in the remaining five the evidence decreased. In two of the former regions (chromosomes 6p21 and 17q) the linkage evidence was actually greater in datasets 4 and 5 than in datasets 1–3.

Transmission disequilibrium test
A transmission disequilibrium test (TDT) was performed for the microsatellite markers within the 10 candidate regions with MLODs over 1.0 as well as for the markers on chromosome 17. As expected, the only significantly positive association was with the functional polymorphisms of the HLA DRB1 gene; {chi}2sum,d.f.=14=75.43, P<0.00001. The next most significant value, outside of the MHC, was for D17S789; {chi}2sum,d.f.=9=30.81, P=0.00072. We have previously reported this marker as showing evidence of transmission distortion in a subset of the families (datasets 1–4) presented here (23). In the remaining, independent sample of families (dataset 5) the TDT was replicated with distortion observed in the same direction – an under-transmission of allele 2 (Table 3). As the families in dataset 5 are largely without parents, the parents were reconstructed using only the unaffected siblings. The only other P-value less than 0.05 for the TDT analysis was for D5S392; {chi}2sum,d.f.=15=34.80, P=0.027.


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  		Table 3. Transmission disequilibrium for D17S789
 
Stratification based upon HLA
Stratifying sibling pairs based on the presence/absence of HLA DRB1*15 allowed us to examine potential relationships of other susceptibility loci with HLA. Among the 219 sibling pairs of dataset 1–3, 105 sibling pairs were both positive for the DRB1*15 allele and 56 sibling pairs were both DRB1*15 negative. The genotype data for all 498 markers were re-analyzed for these two subgroups and the MLODs over 1.0 are presented in Table 4. Markers at the MHC are not included in Tables 4 and 5 as the positive findings are inflated due to the stratification. In the DRB1*15 negative families, there were 11 regions with MLODs over 1.0 (more than expected) and in the DRB1*15 positive there were five (as expected). The highest scores were at 11p15 and 10p15 (MLODs of 2.87 and 2.37, respectively) in the non-DRB1*15 bearing sibling pairs (Table 4). The highest MLOD in the DRB1*15 positive sibling pairs was at Xp22 with an MLOD of 1.72 (Table 4).


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  		Table 4. MLODS>=1.0 (in bold) in sample of DRB1*15 positive sibling pairs (n=105) and DRB1*15 negative sibling pairs (n=56)
 

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  		Table 5. The candidate regions stratified by HLA DRB1a status
 
We examined the two groups for sharing and not sharing by creating a normal distribution of the differences between the siblings two-point sharing and not sharing at the respective loci. This analysis is similar to what we described above for ASPs and DSPs. Chromosome 6 markers (n=20) were excluded and the remaining 461 markers were tested. Given the number of markers, we would expect to observe 11.5 Z-scores over 1.96 (0.025*461 markers) by chance; however, 54 markers were observed with Z-scores greater than 1.96, strongly indicating differences between these two groups (Fig. 3). The presence of more linkage evidence in the DRB1*15 negative subgroup is suggestive of genetic heterogeneity between other loci and HLA rather than epistasis (24).



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  		Figure 3. Expected versus observed Z-scores in HLA stratified sample: red line, Z-scores of stratified sample; blue line, expected distribution of Z-scores.

 
The 10 previously identified candidate regions genotyped in the total 552 sibling pairs were also analyzed for the sample divided into those sibling pairs who were both DRB1*15 positive (n=269) or both negative (n=144). The results of these analyses are given in Table 5.

Families with more than two affected siblings
There were 43 nuclear families with more than two affected offsprings (Table 1). These, potentially, may represent a genetically ‘enriched’ sample and were analyzed separately. Of the 11 regions showing evidence for linkage in the combined sample, only the MHC and chromosome 2q37 showed MLOD>1.0 in this subgroup. The MHC marker D6S461 had an MLOD of 3.13 (65.3% sharing) while D2S172, which is 1.45 cM telomeric to D2S2317, had an MLOD score of 2.86 (63.4% sharing). Chromosome 17q22–24 was the only other region with elevated sharing and the MLOD for marker D17S785 was 0.95 (60.1% sharing).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
With few exceptions, the genetic analysis of complex traits has been slow to identify susceptibility genes responsible for predisposition to common diseases (25). It is likely that the inherent genetic complexity of these conditions has been underestimated. Nevertheless there have been some successes. In the case of diabetes mellitus, IDDM1 and IDDM2 were identified as candidates because of their functional relevance (26,27). CTLA-4 has recently been identified as an IDDM susceptibility gene of mild effect by association, TDT and functional studies (28). CTLA-4 was a candidate as it resides in a region previously highlighted by linkage and association findings, in addition to having homology to idd5 of the murine type 1 diabetes model (29–31). Nevertheless it is clear that CTLA-4 cannot account for the previously reported linkage evidence.

Significant linkage to chromosome 16 (32) and, importantly, replication (33) were observed in ASPs with Crohn's disease. This finding, in addition to subsequent linkage disequilibrium studies and knowledge of the pathophysiology of Crohn's disease, led to the identification of the susceptibility gene NOD2/CARD15 (34,35).

Despite overwhelming evidence from genetic epidemiology implicating genetic influences on MS susceptibility, there are still no clearly significant and confirmed non-MHC linkages. Many regions have been highlighted at levels of possible and suggestive significance. Only two studies have met the criteria for significant linkage for loci on chromosomes 19q13, 12q23, 6q27 and 17q22–24 (19,20), but these findings have yet to be confirmed by significant linkage in independent samples.

We report here the largest linkage study in MS. With the exception of the MHC, no multipoint MLOD reached statistical significance (MLOD>3.6). To assess the meaning of a number of positive scores below the threshold of significance, we examined the overall distribution of allele sharing for ASPs compared with what would be expected based simply on chance. Indeed we did observe a shift in the overall distribution of allele sharing towards greater sharing. This shift was not due to excess sharing at a small number of locations. Instead the entire distribution appeared to be shifted towards increased sharing. This result is most consistent with a moderate to large number of susceptibility loci and is similar to the conclusion drawn from a comparable analysis of autism (22). We performed a similar analysis for DSPs (affected–unaffected); we believe this is a robust control since all affected and unaffected siblings were genotyped simultaneously. For the discordant pairs we found no overall excess of allele sharing and the distribution of allele sharing was not shifted towards greater sharing than expected. Although we are able to provide robust evidence for genetic susceptibility, we cannot provide a strong indication of the location of the various genetic contributors, with the exception of the HLA region on chromosome 6p21. However, from the statistical perspective, the various locations we describe are only of suggestive significance.

MHC
Not all groups presenting genome-wide linkage results in MS sibling pairs report significant linkage to the MHC, but all did show elevated sharing with markers at 6p21. This is inevitable given the clear association with the HLA Class II region, although few linkage studies have corrected for the presence of the known association (36). With our relatively dense map and sample numbers, the single significant MLOD for this investigation was observed for MHC Class II HLA DRB1 (MLOD=4.4). The MHC has been known to be associated with MS susceptibility for decades (37,38). The linkage disequilibrium present within the MHC has made the fine mapping of the MS susceptibility gene(s) within the region difficult and the mechanism for the action of HLA antigens on MS risk continues to be incompletely understood.

Region 2q37–tel
In the first stage (datasets 1–3) of our genome scan, no regions met an empirical genome-wide significance level of 0.05 (MLOD=3.37). We were, however, encouraged to genotype further based on the increased evidence for linkage in the total sample compared with expected (Fig. 2). This suggested to us that at least one, or more, of these loci may contain a susceptibility gene and are not just false-positives. Upon re-analysis of the total 552 sibling pairs, the non-MHC region 2q27 met the criteria for suggestive linkage (39) with a final MLOD of 2.27. The suggestive linkage at 2q37–tel is novel. One possible candidate gene in this region is the ATSV gene (axonal transport of synaptic vesicles) and it is supported by neuropathology showing the length-dependent degeneration of selected axons (40).

Region 5p15
Markers on chromosome 5p continued to be positive in our sample. This region was originally implicated in our first genome scan (11) and in more recent Swedish studies (41,42). Other groups (43,44) have also found evidence for a locus on 5p, but the regions were more centromeric than the Canadian region at 5p13–15. Markers in our ‘hotspot’ location have been previously followed-up in datasets 1–4 with three microsatellite markers (23). In the current investigation, we have added dataset 5 to the previous information as well as an additional nine microsatellite markers. The MLODs continued to be modestly positive in dataset 5. In the total sample, the highest MLOD has now shifted telomerically to marker D5S2005 and was maximal at an MLOD of 2.09 and 57.4% sharing.

Region 17q22–24
Another region followed-up in datasets 4 and 5 was 17q, a region highlighted in the British and Finnish whole-genome scans (12,14). The Finnish group has since refined the location of a susceptibility locus to a 4 cM region of 17q22–24 in 22 multiplex families (19). Of note, in our original screen of dataset 1–3, the highest MLOD in the region (MLOD=0.76) did not attain our arbitrary cut-off score of MLOD>=1.0. However, when the chromosome was further genotyped, the MLOD increased to a final score of 1.67; i.e. the fifth highest of our positive findings. Given the positive results reported elsewhere, chromosome 17q warrants fine mapping in the Canadian sample as has been done in the Finnish families. Marker D17S789 at 17q22–24 showed the most transmission distortion outside of the MHC and maps to the Finnish critical region (19). In this study we replicated in dataset 5 our previous linkage disequilibrium results for this marker in datasets 1–4 (23). A comprehensive linkage disequilibrium mapping project with SNPs in functional and regulatory regions of genes is planned and will hopefully illuminate the location of any susceptibility locus present here.

Stratifying by HLA
Our preliminary analysis of the global distribution of allele sharing indicated a shift towards excess sharing among ASPs versus DSPs. We repeated this analysis on subsets of sibling pairs stratified based on the absence/presence of DRB1*15. As described by Risch (24) a model of loci having a multiplicative relationship with DRB1*15 would predict no difference in the sharing distribution for these two subsets. A model of positive interaction, stronger than a multiplicative interaction, would predict more sharing in the DRB1*15 positive sibling pairs, while a model of genetic heterogeneity or an additive model predicts greater sharing in the DRB1*15 negative sibling pairs. Our results from this subset analysis were clear-cut. The DRB1*15 positive subgroup showed no excess of allele sharing at all for non-chromosome 6 loci, similar to what we observed for DSPs. On the other hand, the DRB1*15 negative subgroup showed significant excess allele sharing outside chromosome 6p. Among 461 markers tested, 54 showed a significant excess of sharing (at the 0.025 level) in the DRB1*15 negative subgroup, far exceeding the 11.5 expected. Thus it appears that the excess allele sharing we observed in the total sample of ASPs was due to increased sharing within the DRB1*15 negative subgroup, and that the excess is due to moderate to large rather than a small number of loci.

As was true for the entire sample of sibling pairs (except 6p21) we also did not obtain any formally significant findings within either DRB1*15 defined subgroup, although several regions achieved the level of suggestive linkage.

We did not confirm the positive findings presented in a UK study of 227 sibling pairs at 1p, 17p or X in the DRB1*15 positive sibling pairs or at 1cen, 3p, 7p, 14q or 22q in the DRB1*15 negative sibling pairs (45). Similarly, we did not confirm evidence for linkage observed in a American study at 13q33 in the DRB1*15 positive families. However, we did observe some evidence for linkage (MLOD=1.07) to 7q21–22 in the DRB1*15 negative families (Table 3) as also reported by the American group (20). We observed evidence for linkage at 11p13–15 (MLOD=2.87), 10p15 (MLOD=2.37) in the DRB1*15 negative families and an MLOD of 1.72 at Xp22 in the DRB1*15 positive families. In the combined sample of 552 sibling pairs, evidence for linkage at 18p11, 9q21 and 21q22 seems to be restricted to the DRB1*15 bearing sibling pairs while the evidence at 11p13–15 is also present in the DRB1*15 negative findings. A scan of Sardinian MS families also showed evidence for linkage to this region (15) and the Sardinian MS population is largely devoid of the DRB1*15 allele (46).

In summary, we obtained evidence for genetic susceptibility to MS by finding that allele sharing was increased in ASPs, compared with both the expected and the observed distribution for DSPs. However, the evidence significantly supported a moderate to large number of susceptibility alleles. With the exception of chromosome 6p21, no individual location achieved formal statistical significance. In the final analysis of 552 ASPs, the best linkage evidence was obtained at a novel location on chromosome 2q and a previously reported location on chromosome 5p. The next most significant locations are 18p, 9q and 1p. Furthermore, most locations reported as linked in other studies are not seen in our Canadian sample, with the possible exception of chromosome 17q. Here we have both a moderately positive MLOD score and replicated evidence of association by TDT analysis. Analysis stratified based on the presence/absenceof DRB1*15 showed that the overall excess allele sharing was restricted to the DRB1*15 negative group but, again, no formally significant LOD scores were obtained for any specific location.

Our findings provide some guidance regarding future directions for genetic analysis. Additional genes remain to be found and chromosomes 2q, 5p and 17q appear likely candidate regions for additional linkage and disequilibrium mapping. Also, the susceptibility genes appear to be enriched in the DRB1*15 negative families and future studies should focus attention on this subgroup.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
The collection of the Canadian MS family material has been described in detail elsewhere (47) but represents the accumulation of 20 000 patients by a network of MS clinics begun in 1982 (48). Probands and the affected siblings were classified as ‘affected’ if they met the criteria for clinically definite MS as defined by Poser et al. (49). The family material was arranged, historically, into ‘datasets’ as follows: (i) dataset 1, 97 sibling pairs from 58 families; (ii) dataset 2, 44 sibling pairs from 42 families; (iii) dataset 3, 78 sibling pairs from 72 families; (iv) dataset 4, 114 sibling pairs from 97 families; (v) dataset 5, 219 sibling pairs from 178 families (11,23). The characteristics of these families are summarized in Table 1. Datasets 1–3 consist of 172 families and 219 sibling pairs and were considered as the first stage of the genome analysis. Datasets 4 and 5 consist of 270 families and 333 sibling pairs and were used to follow-up regions of interest from the first analysis. All families were of Caucasian descent. In total 1978 individuals were genotyped.

Marker map
In our original genome scan of dataset 1, 257 microsatellite markers with an average spacing of 15.2 cM were used (11). We have now added another 241 markers, chosen from the CHLC, Genethon and Marshfield databases, in addition to our original 257 markers (n=498), for an average spacing of 7 cM.

For this investigation, the additional 257 markers were genotyped in dataset 1 and the total of 498 markers was genotyped in datasets 2 and 3. The markers typed in datasets 4 and 5 were those that attained an MLOD score of 1.0 or greater from analysis of datasets 1–3.

Genotyping
Genomic DNA was isolated and purified from peripheral blood samples by standard protocols. Polymerase chain reaction (PCR) was performed using the TC-1600 (Intelligent Automation System) or PTC-225 (MJ Research). PCR conditions for microsatellite genotyping were as follows: final volume of 10 µl with 50 ng of genomic DNA, 10 mM Tris–HCL, pH 8.3, 50 mM KCl, 1.0–3.0 mM MgCl2, 0.60 µmol unlabeled primer, 200 µM of each deoxynucleotide triphosphate and 0.25 U of Taq DNA polymerase. If the marker was to be run radioactively 0.12 µmol [{gamma}-32P]dATP-labeled primer was used. If the markers were to be genotyped fluorescently, primers were end-labeled with FAM, NED or TAMRA, and HEX. Cycle conditions were 94°C for 5 min, 30 cycles of 94°C for 1 min, 50–62°C for 1 min, followed by an elongation step for 5 min at 72°C. Radioactive PCR products were separated by size on a 6% denaturing acrylamide gel (Sequagel-6, National Diagnostics) and separated products were visualized by autoradiography. Gels were exposed to Kodak XAR-5 film for 6–48 h at –70°C. Fluorescent products were separated on ABI 373XL, ABI 377 or ABI 3700 genotyping platforms (Perkin-Elmer/Applied Biosystems). The data from the automated fluorescent platforms was scored using the Genescan 3.1 and the Genotyper software 2.5 (Applied Biosystems). Control samples were included for every 96-well plate in order to ensure consistency in allele calling between plates and between different genotyping methodologies.

HLA DRB1 genotyping
A PCR-based method for typing individuals at the DRB1 locus was performed for this study (50). Sequence-specific primers were used to amplify alleles corresponding to HLA DRB1* 1, 4, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 and 18. The same conditions for the PCR reactions were applied as above with the exception that 100–200 ng genomic DNA was used as template and annealing temperatures ranged between 61 and 66°C. Control primers specific for the third intron of the HLA DRB1 gene were also amplified. PCR products were run on 1.5–2% agarose gels, stained with ethidium bromide and visualized with UV light.

Statistical analysis
Linkage and transmission disequilibrium analysis was performed with the Aspex Statistical Package (ftp://lahmed.stanford.edu/pub/aspex). The sib_ibd and sib_phase programs of the Aspex statistical package were used for a multipoint linkage analysis. Marker distances were taken from the Marshfield research group's ‘build your own map’ function at www.marshfield.org/genetics. The sib_tdt program of the Aspex statistical package was used to test for transmission distortion (51). A locus-counting method was used to derive empirical genome-wide significance values based upon the map density, family members available for genotyping and the completeness of the genotyping (52).


   ACKNOWLEDGEMENTS
 
We would especially like to thank Beverly Scott, Katie Morrison and Angie Shaw for their help with genotyping and data organization. We would also like to thank Irene Yee, Colleen Guimond, and the research staff at the Vancouver Coordinating Center and London Research Center: Kevin Atkins, Randy Holmes, M. Criscuolus, C. Cheung, A. Fok, Steve Polland and Sandra Noble-Topham. The authors are indebted to Calvin Peters for proof reading the manuscript. Informed consent was obtained from all subjects and the experiments performed for this investigation comply with current guidelines and ethics as set by the respective participating MS Clinic Sites of the Canadian Collaborative Study Project. The HLA DRB1 genotyping was performed, in part, by A. Ligers and J. Hillert at the Karolinska Institute (Huddinge, Sweden). This work was funded by the Multiple Sclerosis Society of Canada Scientific Research Foundation. D.A.D. and C.J.W. are funded by the Multiple Sclerosis Society of Canada Research Studentship awards. A.D.S. is a Michael Smith Foundation for Health Research Distinguished Scholar.


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

{dagger} The Canadian Collaborative Study Group is: Vancouver: Donald W. Paty, Stanley Hashimoto, Virginia Devonshire, John Hooge, Lorne Kastrukoff, Joel Oger, Tony Traboulsee; Calgary: Luanne Metz; Edmonton: Sharon Warren; Saskatoon: Walter Hader; Ottawa: Mark Freedman; Kingston: Donald Brunet; Hamilton: John E. Paulseth; London: George Rice, Marcelo Kremenchutzky; Toronto: Paul O'Connor, Marika Hohol, Trevor Gray; Montreal: Pierre Duquette, Yves Lapierre; Quebec City: Jean-Pierre Bouchard; Halifax: T. John Murray, Virender Bhan, Charles Maxner, St Johns: William Pryse-Phillips, Mark Stefanelli. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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