Candidate gene analyses and genome-wide screens in multiple sclerosis
Sammanfattning: Specific disease causing mutations have been identified for many disorders. Most of these disorders are of Mendelian origin and there have been very few successful attempts in the genetic dissection of complex traits. Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the central nervous system with clear influence of both environmental and genetic factors. It is genetically complex, however to date the only genetic region clearly demonstrated to be involved in MS is the major histocompatibility complex. In our studies we have analyzed two NIS candidate genes as well as tried to identify chromosomal regions harboring MS susceptibility genes by genome-wide screens. Alteration in interferon-gamma (IFN-gamma) production has been found in several diseases including multiple sclerosis. Such alterations could theoretically be caused by polymorphisms in the gene. We screened the IFN-gamma gene promoter and part of the first intron for possible mutations by sequencing. We identified a C to T substitution in the IFN-gamma promoter at position -333. Screening for this mutation by sequence specific PCR in 214 MS patients and 164 controls identified two patients, both heterozygous, but no controls with this mutation. The IFN-gamma gene seems to be rather conserved and most probably changes in IFN-gamma expression are due to variations in transcription factor activity, rather than gene polymorphisms. One of the factors influencing IFN-gamma production is interleukin-18 (IL-18). We cloned the human IL-18 promoter and screened it for possible polymorphisms. Five single nucleotide polymorphisms were identified. Three combinations of these polymorphisms were observed in the Swedish population and were analyzed for activity. All alleles had clear promoter activity, which increased after stimulation with PMA/ionomicin. There were no significant differences in promoter activity between alleles before stimulation, but after stimulation, one of the alleles had clearly lower activity than the others. Measurement of IL-18 and IFN-gamma production by RT-PCR showed slightly higher expression of IL-18 in individuals homozygous for the most frequent allele. Two polymorphisms, identifying promoter alleles, were analyzed by sequence specific PCR in MS patients and healthy controls, however no significant differences were found. In the next tree studies we have used isolated and localized Swedish populations to screen the genome for NIS susceptibility regions. By haplotype sharing analysis we studied a genetically isolated population from Överkalix in Northern Sweden, where a high incidence of MS was previously reported. Genealogical analysis had shown that 19 MS patients available for our study, originated from a single common ancestral couple in the eighteenth century. Five affected individuals were selected for an initial genomic screen with 390 microsatellite markers. Shared haplotypes identified in these patients were analyzed in other MS patients and healthy relatives. A conserved haplotype spanning 10 cM was identified on 17p11. Surprisingly, DR-typing revealed no significant sharing of the 14LA region. In the next study we extended the analysis of the Överkalix MS pedigrees and performed a genomewide screen in all available families. We analyzed data by haplotype based transmission disequilibrium test (TDT) and likelihood-ratio test for linkage disequilibrium. Four regions showed significant p-values in both tests. By a haplotype-based TDT analysis, several additional regions were identified. The two most interesting loci were in chromosomes 10p13 and 17p11, being identified by both approaches and corresponding to loci identified in other studies. We also performed a genome-wide screen with 834 microsatellite marker in a family material collected in Värmland county of Sweden, consisting of 54 MS patients and 114 healthy family members. A group of families were possible to track back to common ancestors in the 17th century. We used single marker and haplotype based TDT analysis and nonparametric linkage analysis to analyze data. Regions on chromosomes 2q21-33, 6p25-23, 6q25-27, 14q24-31 and 17q22 were found to be in transmission disequilibrium with MS. Most interesting region was 14q24-31, where several dimarker haplotypes were in transmission disequilibrium in the affected individuals. One marker in this region was also positive in single marker TDT.
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