There is a prevailing hypothesis that multiple sclerosis (MS) is a polygenic immune-mediated disease. So far only one genetic factor has been identified located in human leukocyte antigen (HLA) class II, specifically DR15, DQ6. However, there is no convincing evidence of a common susceptibility locus. We have identified a pedigree of Pennsylvania Dutch extraction, in which MS segregates with an autosomal dominant inheritance pattern. We have collected blood samples from 18 family members, seven of whom show typical signs of MS lesions by MRI. The 18 individuals were serotyped for HLA class I and II and analyzed by a genome-wide screen for linkage analysis. We have found evidence for suggestive linkage to markers on 12p12 with a maximum multipoint lod-score of 2.71 conditional on the presence of DR15, DQ6. Additionally independent studies using mice with chronic relapsing experimental allergic encephalomyelites (CREAE), the mouse model for MS, have shown that there are beneficial effects in multiple sclerosis mediated by cannabinoid receptors. This focus is based on the data obtained in our previous work (Vitale E., Cook S., Sun Specchia C., Subramanian K., Rocchi M., Nathanson D., Schwalb M., Devoto M., and Rohowsky-Kochlan C. Linkage analysis conditional on HLA status in a large North American pedigree supports the presence of a multiple sclerosis susceptibility locus on chromosome 12p12. Human Mol Genetics 2002 3:295-300) , our major objective is to better define the locus on 12p12 as this will further help us in the identification of the gene involved in MS. Our hypothesis is that MS in this family is caused by a two-locus model of inheritance and we are proposing to investigate the 12p12 genomic region for the presence of a gene/s, which mutation is critical for MS susceptibility. In a parallel study using CREAE mice we will begin a study focussed on possible interactions between differentially expressed patterns involved in the determination of MS (Di Marzo V, Bisogno T, De Petrocellis L. Expert Opin Ther Targets. 2001; 5:349-362; Di Marzo V, Bifulco M, De Petrocellis L. Trends Pharmacol Sci. 2000; 21:195-197).
Aim 1. Genetic refinement of the new recognized locus on 12p12. The first step, to refine the critical region, is to use all the markers reported to encompass our region by different databases, which have not yet been characterized.
Aim 2. Gene expression analysis Two systems are currently in use:
1.Commercial (Affymetrix) oligonucleotide arrays - these slides contain 25mer oligonucleotides representing 12,000 human genes. Total RNA extracted from two different sources are reverse transcribed into cDNAs containing the T7 RNA polymerase promoter and hybridized to the oligo array. Quantitation of incorporated label using appropriate scanners and data analysis determines increases, decreases or no change for the 12,000 genes represented on the array.
2. Self printed oligonucleotide arrays - these arrays contain 70mer oligonucleotides, representing 19,000 human genes spotted by automated robot onto polylysine coated glass slides. Total RNAs are directly labeled during the oligo dT cDNA synthesis reaction with either Cy3 or Cy5 and hybridized together to the array. Quantitation of incorporated label is done by fluorescent scanning and use of in house software, to determine relative RNA variations among 19,000 genes.
Focus