We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by ∼30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.The envisioned benefits of high-throughput single nucleotide polymorphism (SNP) analysis are numerous (Brookes 1999), and several large-scale SNP discovery programs are now underway or have been completed (Taillon-Miller et al. 1998;Wang et al. 1998;Cambien et al. 1999;Cargill et al. 1999;Emahazion et al. 1999;Marshall 1999;Picoult-Newberg et al. 1999). Additionally, a number of SNP databases have been built and are steadily growing in content, that is, HGBASE ; http://hgbase.cgr.ki.se), dbSNP (Smigielski et al. 2000; http://www.ncbi.nlm.nih.gov/ SNP) and the SNP Consortium (TSC) (Marshall 1999; http://snp.cshl.org). In order to fully realize the benefits of such developments, further improvements in SNP genotyping technologies will be required. Critical issues here will include ease of assay design, equipment complexity, assay cost, reliability, accuracy, flexibility, and compatibility with automation. Alternative methods under development in different laboratories possess various advantages and disadvantages, making each suitable for a different range of applications. Arguably, however, standardized and simple assay design in addition to accurate allele determination are perhaps the most important prerequisites for a broadly applicable method. Given these features, further development efforts would enable ...

Although familial PNE probably arises from polygenic inheritance, analysis of a large number of families may enable the identification of major susceptibility loci. Recently, linkage analysis with polymorphic markers identified a locus predisposing to PNE, assigned ENUR1, in a proportion of Danish families.'4The gene resides in a 4 cM interval flanked by markers D13S263 and D13S291 on chromosome 13q.In a clinical and genetic study we examined two aspects of PNE. First, we investigated the genetic predisposition to PNE in 392 cases by recording family history of enuresis. Second, we performed linkage analysis in 16 families segregating for dominant PNE. Materials and methods PATIENTSIn a continuing Swedish multicentre study," 392 children (291 boys and 101 girls) older than 6 years were identified. All showed a severe form of PNE including .10 wet nights out of 28 and absence of a dry period >3 months. Concomitant neurological or urological dysfunction associated with PNE was excluded. Evaluation of each patient included physical examination and urine analysis.A detailed family history was recorded in each case in order to determine the presence of familial PNE as defined by any close relative with PNE beyond the age of 6 years. The parents of affected probands underwent a structured interview with specific questions regarding the onset, frequency, and cessation of bedwetting. Direct contact was made with the relatives when the information from the parents indicated a positive family history for PNE. The diagnosis among second and third degree relatives was ascertained, or excluded, with the same criteria as for the proband. Fam-ily members under the age of 7 years were not included.

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Multiple sclerosis (MS) is a T-cell-mediated disease of the central nervous system, characterized by damage to myelin and axons, resulting in progressive neurological disability. Genes may influence susceptibility to MS, but results of association studies are inconsistent, aside from the identification of HLA class II haplotypes. Whole-genome linkage screens in MS have both confirmed the importance of the HLA region and uncovered non-HLA loci that may harbor susceptibility genes. In this twostage analysis, we determined genotypes, in up to 672 MS patients and 672 controls, for 123 single-nucleotide polymorphisms (SNPs) in 66 genes. Genes were chosen based on their chromosomal positions or biological functions. In stage one, 22 genes contained at least one SNP for which the carriage rate for one allele differed significantly (Po0.08) between patients and controls. After additional genotyping in stage two, two genes-each containing at least three significantly (Po0.05) associated SNPs-conferred susceptibility to MS: LAG3 on chromosome 12p13, and IL7R on 5p13. LAG3 inhibits activated T cells, while IL7R is necessary for the maturation of T and B cells. These results imply that germline allelic variation in genes involved in immune homeostasis-and, by extension, derangement of immune homeostasis-influence the risk of MS.

The genetic susceptibility to collagen-induced arthritis (CIA) in mice, the most commonly used model for rheumatoid arthritis, has been analyzed. The highly susceptible B10.RIII strain was crossed with the resistant RIIIS/J strain and the F2 intercross mice were subjected to genomic screening using microsatellite markers. These strains share the MHC region on chromosome 17, known to be of importance in CIA (this locus is named Mcia1). The same cross has earlier been used to map the major genes outside the MHC controlling chronic relapsing experimental allergic encephalomyelitis (EAE). It was found that the major locus controlling CIA (Mcia2; lod 4.12) was located on chromosome 3 in the same region as one of the major loci controlling EAE (Eae3). The linkage was reproduced in a mouse strain in which the locus was isolated on the B10.RIII background at the N4I2 generation. A second putative locus was identified on chromosome 13 (lod 3.13). The present finding identifies new loci outside the MHC controlling CIA and provides evidence that mouse CIA is controlled by polymorphic genes.