Robert Feakes

HLA typing in the United Kingdom multiple sclerosis genome screen.

ABSTRACT The United Kingdom multiple sclerosis genome screen demonstrated a peak maximum lod score of 2.8 in the HLA region, together with statistically significant excess transmission of the 121-base pair (bp) allele of the tumour necrosis factor-a marker. In order to determine whether this association is independent of the established HLA association, or simply a consequence of the 121-bp allele being part of the same haplotype, we HLA-DR and -DQ typed the 227 sibling-pair families used in the original screen. The expected associations of multiple sclerosis with the DR15 (p=8.7E-18), DQ6 (p=2.0E-09) and DR51 (p=2.8E-16) phenotypes were confirmed, and excess transmission of the DRB1*1501 and DQB1*0602 alleles was demonstrated. Combining HLA typing with the original microsatellite data demonstrated extensive linkage disequilibrium between the 121-bp allele and the 1501-0602 haplotype. Outside this extended haplotype (121-1501-0602), none of the alleles demonstrated significant transmission distortion. Having established the importance of this extended haplotype, we reanalysed the entire genome screen data after excluding those sibling pairs sharing the extended haplotype (n=27). Conditioning the full genome screen data on the basis of identity by state sharing showed that some potential linkage regions identified in the original screen clustered in families, in which the extended haplotype was shared (1p, 2p and 17q), whereas others grouped with those in which it was not (5cen, 7p and Xq). This suggests complexity in the genetics of multiple sclerosis.

A SCREEN OF CANDIDATES FROM PEAKS OF LINKAGE : EVIDENCE FOR THE INVOLVEMENT OF MYELOPEROXIDASE IN MULTIPLE SCLEROSIS

We tested 11 microsatellite markers for evidence of transmission distortion in 744 trio families with multiple sclerosis. Ten of the markers lie within or near to candidate genes selected on the basis that they map within the regions of potential linkage identified in our previously reported linkage genome screen, while the eleventh is an anonymous marker which had previously shown modest evidence for transmission distortion in our sibling pair families. Only the marker related to the myeloperoxidase (MPO) gene revealed tentative evidence for linkage disequilibrium and further work on this gene is clearly needed in order to resolve the status of this region in conferring susceptibility to multiple sclerosis.

Interleukin 1 receptor antagonist (IL-1ra) in multiple sclerosis

The autoimmune nature of multiple sclerosis introduces cytokine genes as logical candidates for the loci determining susceptibility to the disease, and/or influencing disease progression. Working on this principle, several groups have investigated the relevance of polymorphism in the interleukin 1 receptor antagonist gene (IL1RN) but with conflicting results. In an effort to clarify this situation, we typed the functionally significant variable number of tandem repeat (VNTR) polymorphism from intron 2 of IL1RN in 536 simplex families with multiple sclerosis. In order to improve the information extracted from these families, we also typed a closely mapped single nucleotide polymorphism (SNP) from the promoter of IL1B (the gene for IL-1beta). Disease associations were assessed by transmission disequilibrium testing (TDT), alone and after haplotype construction. There was highly significant (P</=2.48.10(-16)) linkage disequilibrium (LD) between the two polymorphisms studied, illustrating that LD adjacent to an SNP can be considerably more extensive than has recently been suggested. None of the alleles from the VNTR, the SNP or their haplotype showed statistically significant evidence for association. We stratified patients for current disability status but using this manoeuvre found no evidence that either of the polymorphisms influences disease severity. Combining the available data on the IL1RN VNTR suggests that any effect of this gene on susceptibility to multiple sclerosis, or its progression is, at best, small.

Exploring the dense mapping of a region of potential linkage in complex disease: an example in multiple sclerosis.

In 1996 we reported the results of a genome screen in multiple sclerosis, in which potential linkage was identified in a total of twenty regions, including the centromeric region of chromosome 5. In order to investigate the efficiency of typing dense arrays of markers in regions of potential linkage, we have typed an additional nineteen microsatellite markers from this chromosome 5 region (D5S623 ‐ D5S428) in the same sibling pair families. The mean additional information extracted per marker typed declined with increasing map density, while inaccuracies in the mapping and the density of genotyping errors increased. Our empirical results suggest that, in linkage‐based experiments, there is a limit to the benefits that are gained from typing additional markers in the same families. Increasing map density up to the 2.5–5 cM level efficiently extracts valuable extra information; however, beyond this level efficiency declines while the confounding effects of mapping and genotyping errors accumulate. We, therefore, recommend that extra markers typed in linkage studies be limited to this level of resolution. Mapping regions beyond this density should only be initiated when searching for linkage disequilibrium. Genet. Epidemiol. 17:51–63, 1999.

Evidence that allelic variants of the spinocerebellar ataxia type 2 gene influence susceptibility to multiple sclerosis

Expanded CAG trinucleotide repeats are known to be responsible for five of the autosomal dominant spinocerebellar ataxias (SCA1, SCA2, SCA3, SCA6, and SCA7). We have typed each of these repeats in 226 multiple sclerosis sibling pair families. No expanded repeats were seen, indicating an absence of SCA phenocopies in clinically defined familial multiple sclerosis. However, transmission disequilibrium testing for these repeats demonstrated significant excess transmission of the 22 repeat length allele of the SCA2 gene (P=4.4E-06) in multiple sclerosis patients. This observation is consistent with pleiotropic effects of the SCA2 gene, with a non-dynamic mutation/polymorphism contributing epistatically to susceptibility in multiple sclerosis.

No evidence for the involvement of interleukin 2 or the immunoglobulin heavy chain gene cluster in determining genetic susceptibility to multiple sclerosis

Here we report the investigation of two promising candidate multiple sclerosis susceptibility genes. Each is biologically plausible, having a function suggesting possible involvement in the pathogenesis of the disease and positional, having existing linkage evidence supporting its candidature. The two differ, however, in the origin of the supporting linkage evidence. This comes mainly from the analysis of animal models in the case of interleukin 2 (IL-2)1 and from human studies in the case of the immunoglobulin heavy chain gene cluster.2 Interleukin 2 is a cytokine intimately involved with both the function and regulation of the immune system. It has both proinflammatory and anti-inflammatory actions, promoting T cell proliferation during cell mediated immune responses and, conversely, being crucial both for the …

NO EVIDENCE FOR ASSOCIATION OF MULTIPLE SCLEROSIS WITH THE COMPLEMENT FACTORS C6 AND C7

Four genome screens in multiple sclerosis have been completed and each has identified evidence for linkage in the pericentromeric region of chromosome 5. This region encodes a number of candidate genes including those for the complement components C6, C7 and C9. We have used a multiplexed oligoligation assay (OLA) to test single nucleotide polymorphisms (SNPs) from the C6 and C7 genes for evidence of association with multiple sclerosis in our sibling pair families. There was no statistically significant difference in the allele frequencies of these polymorphisms in the index cases from our families when compared with locally derived controls. No evidence for transmission distortion was seen with any of the polymorphisms, or with the haplotype built from the three SNPs from the C7 gene. Despite offering themselves as potential candidates these complement genes appear not to confer susceptibility to multiple sclerosis.

More evidence that founder effects exist in the European population.

In 1996, the chemokine receptor CCR5 was shown to be involved in the process by which the human immunodeficiency virus (HIV) gains entry into CD4 lymphocytes. Shortly after this discovery it was found that inheritance of a naturally occurring 32 base pair (bp) deletion in the receptor’s gene (CMKBR5) significantly reduces the risk of infection in those exposed to HIV, the deletion bearing allele (∆ccr5) coding for a non-functioning protein. These observations raised the possibility that chemokine receptor polymorphisms, such as ∆ccr5, might be important determinants in the development of other retroviral diseases. Although the environmental factor involved in the pathogenesis of multiple sclerosis is uncertain, a novel retrovirus is one candidate. With this in mind, Bennetts et al compared the frequency of the ∆ccr5 allele in Australian patients with multiple sclerosis and unrelated controls, but found no evidence for a protective effect. More recently, Libert et al studied the ∆ccr5 allele in a variety of healthy European populations (not including the UK) and found a striking north–south gradient in allele frequency. In addition, these investigators identified strong linkage disequilibrium (LD) between ∆ccr5 and flanking microsatellite markers suggesting a founder effect with most, if not all, contemporary ∆ccr5 alleles originating from a single ancestral mutation event. In order to establish the role, if any, of ∆ccr5 in patients with multiple sclerosis from the United Kingdom we typed 185 simplex families (both parents and a single affected offspring). The ∆ccr5 allele frequency was 15.4% (57/370) in the 185 affected offspring, with genotype frequencies corresponding to those expected at Hardy Weinberg equilibrium. Transmission disequilibrium testing, performed using the sibtdt program from the ASPEX package version 1.12, revealed no significant evidence for distorted transmission, with 48 ∆ccr5 alleles transmitted and 34 not transmitted (indicating a transmission rate in the 95% confidence interval 47.6%–69.4%). It should be noted, of course, that the modest sample size used in our study means that a small effect from ∆ccr5 cannot be excluded. The flanking dinucleotide microsatellites used by Libert et al (D3S4579 and D3S4580) were also typed in these 185 families; phase of the alleles in each parent was inferred from those transmitted and thereby the frequency of each marker allele in both the ∆ccr5 bearing chromosomes (n = 100) and the wild type chromosomes (n = 640) was calculated (coincidentally the D3S4580 marker was found to have a low frequency null allele, of about 7%). Highly significant linkage disequilibrium was found between ∆ccr5 and the 138 bp allele of D3S4579, and between ∆ccr5 and the 144 bp allele of D3S4580 (see Table). These were confirmed as the same marker alleles found by Libert et al indicating that the UK ∆ccr5 alleles arose from the same common founder. Although we have not found any evidence that ∆ccr5 is involved in determining susceptibility to multiple sclerosis, we confirm the findings of Libert et al. The existence of founder effects in large outbred populations (such as the Europeans) is a significant result in terms of polygenic disease. These data add to the growing evidence supporting the belief that LD frequently occurs in the region of disease genes. In monogenic diseases such as cystic fibrosis the existence