Identification by Denaturing High-Performance Liquid Chromatography of Numerous Polymorphisms in a Candidate Region for Multiple Sclerosis Susceptibility Mara Giordano,* ,1 Peter J. Oefner,† Peter A. Underhill,‡ L. Luca Cavalli Sforza,‡ Roberto Tosi,§ and Patricia Momigliano Richiardi* * Dipartimento di Scienze Mediche, Universita ` di Torino, Novara, Italy; †Department of Biochemistry; and ‡Department of Genetics, Stanford University, Stanford, California 94305; and §Istituto di Biologia Cellulare, CNR, Rome, Italy Received July 27, 1998; accepted December 14, 1998 Genetic association analysis of candidate regions where evidence of linkage has accumulated is becom- ing a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently avail- able single nucleotide polymorphism (SNP) map de- signed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approxi- mately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chro- matography (DHPLC). A systematic search for SNPs was performed in the region 5ptel– q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymor- phisms was performed on 124 sequence tagged sites (STSs) in the 3UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on ex- pressed gene sequences tagged by a set of standard commercially available primers. © 1999 Academic Press INTRODUCTION Testing the association of a trait or disease with unknown genes located in a chromosomal region re- quires the availability of densely spaced polymorphic sites scattered throughout the region of interest. Single nucleotide polymorphisms (SNPs) represent more suit- able genetic markers than microsatellites for this pur- pose since they are highly abundant and less mutating. Moreover SNP genotyping does not require a length measurement of the alleles, and distinguishing alleles can be more readily automated. While recent progress has been reported in SNP development (Wang et al., 1998), the current assemblage of authentic SNP mark- ers remains sparse. A significant acceleration in the detection of biallelic polymorphisms can be achieved by denaturing high-performance liquid chromatography (DHPLC; Oefner and Underhill, 1998). This represents a highly sensitive and automated method for DNA variant detection based on the capability of ion-pair reverse-phase liquid chromatography to resolve homo- duplex from heteroduplex molecules under conditions of partial denaturation. In the present work DHPLC was applied to a sys- tematic search of SNPs on chromosome 5 to obtain a set of biallelic markers to be used in association studies in multiple sclerosis (MS). MS is a relatively common autoimmune disease with a prevalence of about 0.1% in Caucasoids. It is charac- terized by widespread demyelination of the central ner- vous system, leading to neurological disabilities over time. The disease has a substantial genetic component, with 30% concordance in monozygotic twins versus 3% in dizygotic twins and a rather high familial aggrega- tion (Dyment et al., 1997). Although environmental factors certainly have a role in disease susceptibility, familial aggregation is largely controlled by genetic factors as shown by adoption (Ebers et al., 1995) and half-sib (Sadovnick et al., 1996) studies. The current available data suggest the presence of at least one MS locus on human chromosome 5. Data from a complete genome screen in eae mice show strong linkage to a locus, eae2, on a region in the murine chromosome 15 (Sundvall et al., 1995) homologous to human 5p14 – p12. Accordingly, in a study carried out in Finnish multiplex families, a substantial lod score was found with markers on chromosome 5p14 –p12 (Kuokkanen et al., 1996). Finally, data obtained in whole-genome 1 To whom correspondence should be addressed at Dipartimento di Scienze Mediche, Universita ` di Torino, Via Solaroli 17, 28100 No- vara, Italy. Telephone: 39-0321-660606. Fax: 39-0321-620421. E- mail: giordano@med.no.unipmn.it. Genomics 56, 247–253 (1999) Article ID geno.1998.5715, available online at http://www.idealibrary.com on 247 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.