Updated results of the United Kingdom linkage-based genome screen in multiple sclerosis.

In 1996, we reported the results of a linkage genome screen based on 129 UK multiple sclerosis multiplex families, together with follow-up typing of interesting regions in a second set of families. We have now completed screening the remainder of the genome in this second set of United Kingdom families by typing 242 microsatellite markers. These data have been analysed together with those previously published, resulting in the largest currently available whole genome linkage dataset from a single population in multiple sclerosis. Four new regions of potential linkage (chromosomes 10p, 11p, 19p, 20p) not previously described were identified. In the combined analysis of all 226 families, a total of five regions of suggestive linkage are seen (chromosomes 1p, 6p, 14q, 17q, Xq), where only one would have been expected to occur by chance alone.

HLA-DR 15 is associated with female sex and younger age at diagnosis in multiple sclerosis.

BACKGROUND: The association between multiple sclerosis and class II alleles of the major histocompatibility complex, in particular the DRB1*1501-DQB1*0602 haplotype, is well established but their role in determining specific features of this clinically heterogeneous disease is unknown as few studies involving large sample sizes have been performed. METHODS: 729 patients with multiple sclerosis were typed for the HLA DR15 phenotype. All patients underwent clinical assessment and a detailed evaluation of their clinical records was undertaken. RESULTS: The presence of DR15 was associated with younger age at diagnosis and female sex but there was no association with disease course (relapsing-remitting or secondary progressive v primary progressive type), disease outcome, specific clinical features (opticospinal v disseminated form), diagnostic certainty (clinically and laboratory supported definite v clinically probable multiple sclerosis), and paraclinical investigations including the presence of oligoclonal bands in the CSF or characteristic abnormalities on MRI imaging of the central nervous system. CONCLUSION: Even though DR15 carriers are more likely to be female and prone to an earlier disease onset, the results indicate that there is no association with other specific clinical outcomes or laboratory indices examined here. This suggests that DR15 exerts a susceptibility rather than disease modifying effect in multiple sclerosis.

A genome screen for multiple sclerosis in Sardinian multiplex families.

The prevalence of multiple sclerosis in Sardinia is significantly higher than in neighbouring Mediterranean countries, suggesting that the isolated growth of the population has concentrated genetic factors which increase susceptibility to the disease. The distinct HLA association of multiple sclerosis in Sardinia supports this interpretation. We have performed a whole genome screen for linkage in 49 Sardinian multiplex families using 327 markers. Non parametric linkage analysis of these data reveal suggestive linkage in the region of Chr 1q31, Chr 10q23 and Chr 11p15.

A genome screen for multiple sclerosis in Italian families.

We have screened the whole genome for linkage in 40 Italian multiplex families with multiple sclerosis using 322 markers. The GENEHUNTER-PLUS program was used to analyse these data and revealed eight regions of potential linkage where the lod score exceeds the nominal 5% significance level (0.7). No region of linkage with genome-wide significance was identified and none of the markers showed evidence of statistically significant transmission disequilibrium. Overall these results have refined the linkage data relating to this disease in Italians modestly supporting some previously identified areas of interest and helping to exclude others.

No association between multiple sclerosis and the Notch3 gene responsible for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL).

The clinical and radiological overlap between multiple sclerosis and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL; MIM 125310) raises the possibility of diagnostic confusion and suggests that pleiotropic effects of the Notch3 gene might include influencing susceptibility to multiple sclerosis. To investigate these possibilities three microsatellites markers closely flanking the Notch 3 gene in 745 simplex families with multiple sclerosis were genotyped and exon 3 and exon 4 of the gene were directly sequenced in a subset of the index members from these families (n=93). No evidence for association was found in any of the three markers and none of the commoner mutations causing CADASIL were found in the sequenced patients.

Suggestive evidence for association of human chromosome 18q12-q21 and its orthologue on rat and mouse chromosome 18 with several autoimmune diseases.

Some immune system disorders, such as type 1 diabetes, multiple sclerosis (MS), and rheumatoid arthritis (RA), share common features: the presence of autoantibodies and self-reactive T-cells, and a genetic association with the major histocompatibility complex. We have previously published evidence, from 1,708 families, for linkage and association of a haplotype of three markers in the D18S487 region of chromosome 18q21 with type 1 diabetes. Here, the three markers were typed in an independent set of 627 families and, although there was evidence for linkage (maximum logarithm of odds score [MLS] = 1.2; P = 0.02), no association was detected. Further linkage analysis revealed suggestive evidence for linkage of chromosome 18q21 to type 1 diabetes in 882 multiplex families (MLS = 2.2; lambdas = 1.2; P = 0.001), and by meta-analysis the orthologous region (also on chromosome 18) is linked to diabetes in rodents (P = 9 x 10(-4)). By meta-analysis, both human chromosome 18q12-q21 and the rodent orthologous region show positive evidence for linkage to an autoimmune phenotype (P = 0.004 and 2 x 10(-8), respectively, empirical P = 0.01 and 2 x 10(-4), respectively). In the diabetes-linked region of chromosome 18q12-q21, a candidate gene, deleted in colorectal carcinoma (DCC), was tested for association with human autoimmunity in 3,380 families with type 1 diabetes, MS, and RA. A haplotype ("2-10") of two newly characterized microsatellite markers within DCC showed evidence for association with autoimmunity (P = 5 x 10(-6)). Collectively, these data suggest that a locus (or loci) exists on human chromosome 18q12-q21 that influences multiple autoimmune diseases and that this association might be conserved between species.

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

Pulsed monoclonal antibody treatment and autoimmune thyroid disease in multiple sclerosis.

BACKGROUND: Multiple sclerosis results from T-cell-dependent inflammatory demyelination of the central nervous system. Our objective was long-term suppression of inflammation with short-term monoclonal antibody treatment. METHODS: We depleted 95% of circulating lymphocytes in 27 patients with multiple sclerosis by means of a 5-day pulse of the humanised anti-CD52 monoclonal antibody, Campath-1H. Clinical and haematological consequences of T-cell depletion, and in-vitro responses of patients' peripheral-blood mononuclear cells were analysed serially for 18 months after treatment. FINDINGS: Radiological and clinical markers of disease activity were significantly decreased for at least 18 months after treatment. However, a third of patients developed antibodies against the thyrotropin receptor and carbimazole-responsive autoimmune hyperthyroidism. The depleted peripheral lymphocyte pool was reconstituted with cells that had decreased mitogen-induced proliferation and interferon gamma secretion in vitro. INTERPRETATION: Campath-1H causes the immune response to change from the Th1 phenotype, suppressing multiple sclerosis disease activity, but permitting the generation of antibody-mediated thyroid autoimmunity.

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.

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.

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.

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.

The genetics of multiple sclerosis: principles, background and updated results of the United Kingdom systematic genome screen.

Genetic susceptibility to multiple sclerosis is implicated on the basis of classical family studies and phenotype analyses. The only reproducible legacy from the candidate gene approach has been the discovery of population associations with alleles of the major histocompatibility complex. Systematic genome scanning has since been applied using a panel of anonymous markers to identify areas of linkage in co-affected siblings. Here, we describe the principles of genome screening and update the UK survey of multiple sclerosis. This identified 20 regions of potential interest, but in none was there unequivocal linkage. In theory, attempting to replicate these findings in a second set of sibling pair families is the most appropriate way to distinguish true from false positives, but unfortunately the number of families required to do this reliably is prohibitively large. We used three approaches to increase the definition achieved by the screen: (i) the number of sibling pairs typed in an identified region of potential linkage was extended; (ii) the information extraction was increased in an identified region; and (iii) a search was made for missed regions of potential linkage. Each of these approaches has considerable limitations. A chromosome-by-chromosome account is given to direct future searches. Although an additional marker placed distal to the 'hit' on chromosome 14q increased linkage in this area, and typing extra sibling pairs increased linkage on chromosomes 6p and 17q, evidence for linkage was more commonly reduced and no additional regions of interest were found. A further refinement of the genome screen was undertaken by conditioning for the presence of HLA-DR15. This produced a surprising degree of segregation among the regions of interest, which divided into two distinct groups depending on DR15 sharing: the DR15-sharing cohort comprised loci on chromosomal areas 1p, 17q and X; and the DR15-non-sharing cohort was made up of loci on 1cen, 3p, 7p, 14q and 22q. This result further highlights the genetic complexity of multiple sclerosis. What can now be inferred is that a gene of major effect is excluded from 95% of the genome and one with a moderate role from 65%, whereas genes which make a very small biological contribution cannot be discounted from any region. The available results suggest that multiple sclerosis depends on independent or epistatic effects of several genes each with small individual effects, rather than a very few genes of major biological importance.

HLA associations with multiple sclerosis in the Canary Islands.

The study of small island populations has proved informative with respect to the epidemiology and genetics of many complex traits including multiple sclerosis. The class II major histocompatibility antigen DR15 is associated with multiple sclerosis in all groups except Sardinians, where the primary association is with DR4. We compared HLA-DR and -DQ allele frequencies in a representative sample of patients with multiple sclerosis from the Canary Islands with appropriate controls. There was a significant association with DR15 (patients 21/53: 40%: controls 11/55; 20%: chi2=4.09; pc=0.04; relative risk [RR]=1.98). DRB1*1501-DRB5*0101 was present in 17/53 (32%) patients in whom sub-types could be identified compared with 6/55 (11%) controls (chi2=7.21; pc=< 0.01; RR=2.94). All DR15 positive controls carried the DQA1*0102, DQB1*0602 haplotype whereas this was only present in 26/30 patients, suggesting that the primary association is with HLA-DR and not -DQ. We also found a significant increase in HLA-DR4 (16/53 [30%] in patients compared with 7/55 [13%] in controls; pc=0.05). This study contributes a new point on the immunogenetic map of multiple sclerosis in Europe, confirming the primary DR15 association with multiple sclerosis in a previously unstudied population but again highlighting the importance of DR4 in Mediterranean peoples.

HLA typing in the United Kingdom multiple sclerosis genome screen.

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.

Hormonal influences on brain ageing quality: focus on corticotropin releasing hormone-, vasopressin- and oxytocin-immunoreactive neurones in the human brain.

In this paper we have investigated the distribution of corticotropin releasing hormone (CRH)-, vasopressin- and oxytocin-immunoreactive (IR) neurones in the paraventricular nucleus in the senile compared to the adult human brain. We found a higher number of CRH-IR neurones in senile compared to adult subjects. Vasopressin- and oxytocin-IR neurones were instead more weakly stained in the former compared to latter. These results support a hypothalamic involvement in promoting the higher activity of the hypothalamus-pituitary-adrenal axis and, thus, higher glucocorticoid plasma levels which have been described in the elderly.