Enthusiasm Mixed with Scepticism About Single-Nucleotide Polymorphism Markers for Dissecting Complex Disorders

MEETING REPORT

First International SNP Meeting at Skokloster, Sweden, August 1998 Enthusiasm mixed with scepticism about single-nucleotide polymorphism markers for dissecting complex disorders

Ann-Christine Syv˚anen1, Ulf Landegren2, Anders Isaksson2, Ulf Gyllensten2 and Anthony Brookes2

1Department of Medical Sciences; 2Department of Genetics and Pathology; Uppsala University, Uppsala, Sweden

Single nucleotide polymorphisms (SNP) are frequent in number of participants, created a relaxed atmosphere our genomes, occurring on average once every thou- that stimulated many informal scientific discussions. sand nucleotides. They are useful as genetic markers Following an introductory lecture from Gert-Jan van because SNPs evolve slowly and because they can be Ommen (Leiden, NL), President of HUGO, there was scored by technically simple methods. Moreover, a a lively discussion on the role of HUGO in the genome great deal of the functional variation that explains project, revolving around its part in linking the diverse human diversity probably results from SNPs. In recent activities in the field, ranging from facilitating mapping years, SNP-based strategies to explain common disease and mutation analysis to nomenclature supervision, have received enormous attention from the scientific providing guidance in genome diversity and population community as well as from pharmaceutical and bio- studies and more generally in the ethical and intellec- technology companies. The enthusiasm stems largely tual property areas. During the two and a half days of from the hope that large-scale association studies using the meeting advances in technology for discovering and SNPs will help identify genes that contribute to scoring SNPs, SNP-based approaches to dissect com- common disorders with complex genetic causation, and plex diseases and the utility of SNPs in the study of allow formulation of improved therapeutic drugs. human populations as well as in bioinformatics, compu- The 1st International Meeting on Single Nucleotide tational, ethical and commercial issues related to SNPs Polymorphism and Complex Genome Analysis was held were discussed. at Skokloster in the heart of Sweden, 29 August–1 September, 1998, with the support of the Swedish SNP Discovery and Screening Foundation for Strategic Research, Cell Factory and Genome Research Programmes, and by the Human Methods Genome Organization (HUGO), through a contract Many large-scale efforts are in progress to discover (BMH4-CT97-2031) between HUGO Europe and the novel SNPs by resequencing genomic regions from European Commission. The meeting convened leading several individuals. Genset (Martha Blumenfeld, scientists in fields related to SNP research. Altogether France) expects to find by 1999 three polymorphisms around 100 scientists, representing both academic from each of 20000 ordered BAC clones, spanning the institutions and companies in 17 different countries, human genome. Similarly, the whole-genome sequenc- were accommodated at the conference facilities at the ing effort at Celera (Mark Adams, MD, USA) will Skokloster castle on lake M˚alaren, out of twice that generate hundreds of thousands of polymorphisms, number of applicants. The remote and peaceful rural although it is at present unclear whether these will be setting of the meeting, together with the relatively small made publicly available. Andrew Clark (Penn State SNPs in dissecting complex disorders A-C Syv˚anen et al t 99

University, PA, USA) and his collaborators have array padlock probes on planar supports, and to target sequenced the LPL gene locus from 24 individuals multiple SNPs along DNA fibres for direct detection of representing three different populations, and they haplotypes of the markers. intend to sequence another 15 candidates to find SNPs that may be informative in the search for cardiovas- cular disease genes. Peter Underhill (Stanford Uni- versity, CA, USA) described the usefulness of denatur- ing HPLC for identifying previously unknown SNPs SNP Databases through heteroduplex analysis, and he also presented Improved bioinformatics tools and public databases the technique as a tool for scoring known SNPs. Several cataloguing SNP variation are of central importance to other approaches to accurate and cost-effective scoring the field. The HUGO mutation database initiative was of SNPs were presented. Microplate array diagonal gel presented by Richard Cotton (Mutation Research electrophoresis (MADGE) represents a simple but Centre, Melbourne, Australia), and Charles Scriver efficient multiplex genotyping assay (Ian Day, South- (McGill University, Canada) described the database for ampton University, UK). Multiplex typing on DNA the phenylalanine hydroxylase (PAH), as a model of a arrays, either through minisequencing (Ann-Christine database serving both the scientific community, clini- Syv˚anen, Uppsala University, Sweden, and Andres cians, and phenylketonuria patients. Elements needed Metspalu, Tartu University, Estonia), or via a ligase fully to describe sequence variants are being worked assay (Ed Southern, Oxford University, UK) were out in conjunction with the sequence variation database shown to distinguish SNPs in heterozygote and homo- at European Bioinformatics Institute (EBI), with the zygote form more accurately than hybridisation-based aim of harmonising database design and ensure accessi- methods. Nonetheless, hybridisation to high density bility (Heikki Lehv˚aslaiho, EMBL-EBI, UK). DNA chips from Affymetrix was shown to be useful for Four major public SNP databases were presented or analysing HLA polymorphism (Henry Erlich, Roche referred to: Molecular Systems, CA, USA), as well as for discover- (i) HGBASE (http://hgbase.interactiva.de) from ing SNPs and typing thousands of them after multiplex Uppsala University and Interactiva Biotechnolo- PCR (Jian-Bing Fan, Affymetrix, CA, and Michelle gie GmbH links more than 2400 SNPs to genes Cargill, Whitehead Institute, MA, USA). and is designed as a purpose-built tool to facilitate As alternatives to the DNA microarray formats, association studies; SNPs can be efficiently scored in individual amplification reactions by enzyme-assisted homogeneous assays (ii) dbSNP (http://www.ncbi.nlm.nih.gov/SNP/ based on fluorescence resonance energy transfer snp.how — to — submit.html), a joint effort by the (FRET) or fluorescence polarisation (Pui-Yan Kwok, NHGRI and the NCBI, is currently accepting Washington University, MO, USA), using the homoge- submissions. It can be searched via sequence- neous, FRET-based TaqMan assay (Ken Livak, Perkin- tagged site (STS) number and will be integrated Elmer/Applied Biosystems, CA, USA), by pyrose- with Genbank; quencing (PDl NyrJn, Royal Institute of Technology, (iii) the SNP Databases from Washington University Sweden), or by dynamic allele-specific hybridisation and the Stanford Genome Centre (http://www.ibc (DASH) (Anthony Brookes, Uppsala University, Swe- .wustl.edu/SNP/) contain several hundred SNPs, den). Throughput in currently available scoring meth- which are being integrated into dbSNP; ods is limited by the difficulty of multiplexing PCR, necessitating large numbers of amplification reactions. (iv) The Whitehead SNP Database (http://www.geno- These contribute significantly to the costs of current me.wi.mit.edu/SNP/human/index.html) contains genotyping assays, as pointed out by James Weber over 3000 SNPs, most of which are chromoso- (Marshfield Medical Research Foundation, WI, USA). mally mapped. The database is searchable by genomic region or STS number. Intramolecular ligation probes – padlockprobes – com- bined with rolling circle replication for signal amplifica- Owing to the anticipated large commercial use of tion offer the promise of SNP scoring in genomic DNA SNPs, most discovered SNPs are currently stored in without prior target amplification (Ulf Landegren, private databases owned by genomics companies or the Uppsala University, Sweden). Work is in progress to pharmaceutical industry. In principle, SNPs can be SNPs in dissecting complex disorders t A-C Syv˚anen et al 100

patented, especially if their relevance to a disease is collect patient samples in order to use the SNPs for disclosed, and many patent applications on SNPs have identifying new drug targets and to define in greater been filed, but as yet no patents have been granted detail the genetic variation that determines the (Christian Stein, Patent and Licensing Agency for the responses to drugs. So far the polymorphisms of the German human genome project). Without patent pro- drug metabolising enzymes of the CYP gene family are tection, a large part of the identified SNPs will no doubt the most thoroughly studied example of pharmacoge- remain in commercial databases that are not publicly netic variation (Magnus Ingelman-Sundberg, Karo- accessible. linska Institute, Sweden). In the most intensely debated contribution at the meeting, Joe Terwilliger (Columbia University, NY, USA) presented his sceptical view of the general utility SNPs and Complex Disorders of SNPs in association studies for finding genes behind complex disorders. He cautioned that over-enthusiastic So far successful association studies for identifying SNP researchers underestimate the complexity of the genes underlying complex traits have exploited SNPs human genome and assume far too low allelic heteroge- within candidate genes, selected on the basis of neity for genes causing complex disorder. His view functional clues. The clear association of the variant received support from Rosalind Harding (John Rad- apolipoprotein allele E4 with Alzheimer disease exem- cliffe Hospital, Oxford University, UK) who failed to plifies successful application of this approach, and other identify the mutant allele causing sickle cell anaemia well known examples are the role of certain HLA using SNP data alone in a model study involving a alleles in diabetes or other autoimmune diseases, as random population sample, and from Andrew Clark discussed by Henry Erlich (Roche Molecular Systems, (Penn State University, PA, USA), who illustrated the CA, USA). In other cases, such as in Parkinson disease, complex pattern of haplotype variability in the lipopro- genetic associations of borderline significance have tein lipase (LPL) gene, due to frequent recombination been found (Nobutaka Hattori, Juntendo University within the LPL locus. School of Medicine, Japan). The chances of finding genes underlying complex diseases may be increased by investigating SNPs in genes that encode the compo- nents of complete metabolic pathways, selected Population Genetics because of suspected relevance to the disease, as exemplified by genes involved in oxidative phosphor- A rapidly growing number of SNPs are being identified ylation in Alzheimer’s disease (Anthony Brookes, in the non-recombining part of the Y chromosome Uppsala University, Sweden). Nancy Pedersen (Kar- (Peter Underhill, Stanford University, CA, USA). olinska Institute, Sweden) recommended combining Haplotypes composed of SNPs and microsatellite association studies using SNPs with established epide- markers on the paternally transmitted Y chromosome miological practices, for example by analysing samples complement the maternally transmitted mitochondrial from the extensive twin registries available in the DNA variation and increase the accuracy of studies on Scandinavian countries. human evolution and population structures (Chris An important explanation for the intense interest in Tyler-Smith, University of Oxford, UK). Population SNPs is the expectation that genome-wide association genetic studies also provide information on the age, studies will serve to identify previously unknown frequency, and distribution of SNPs in a population. genetic variants of importance in complex disorders. This information could be used in the design and Nicholas Schork (Case Western Reserve University, statistical interpretation of the results of SNP-based OH) described a positional SNP strategy to locate disease association studies, to reduce the problems of genes associated with complex traits that is based on allelic heterogeneity. An example of this possibility was calculating the regional strength of linkage disequilib- given by Svante P˚a˚abo (University of Munich, Ger- rium and identification of haplotypes. As reviewed by many) who suggested that linkage disequilibrium cre- Nigel Spurr (SmithKline Beecham, UK) the pharma- ated by genetic drift, rather than by a population ceutical industry has initiated pharmacogenomics pro- bottleneck, could be of value to define genomic regions grammes to identify SNPs, develop statistical tools, and containing complex disease genes. SNPs in dissecting complex disorders A-C Syv˚anen et al t 101 Ethical Considerations Conclusions Sandy Thomas (Nuffield Council on Bioethics, UK) The meeting left little doubt that knowledge about drew attention to the fact that most ethical questions human genetic variation will provide useful tools for related to genetic information discussed so far, such as dissecting the genetics behind complex diseases. A confidentiality, genetic testing and ownership issues, number of more specific questions remain contentious, deal with single gene disorders. The complex genetic however, including how to ensure that information background of multifactorial diseases will raise new about human genetic variation is made widely availa- ethical issues, for which single gene diseases will not ble, how best to ascertain such markers in large patient provide useful models. It is urgent that satisfactory cohorts and, figuring prominently at this conference, ethical guidelines are worked out also for the common how best to design studies in order to avoid them complex disorders, so that the results of the intense generating overwhelming statistical noise, from which it research on human genetic variation using SNPs can be will be hard or impossible to identify significant genetic fully used for the benefit of more targeted and variants. preventive health care in the future.