HUMAN MUTATION 28(5), 441^450, 2007 RESEARCH ARTICLE Patterns of Variation in DNA Segments Upstream of Transcription Start Sites Damian Labuda,1,2 Catherine Labb´e,1 Sylvie Langlois,1 Jean-Francois Lefebvre,1 Virginie Freytag,1 Claudia Moreau,1 Jakub Sawicki,1 Patrick Beaulieu,1 Tomi Pastinen,3 Thomas J. Hudson,3 and Daniel Sinnett1,2 1Centre de Recherche, Hˆopital Sainte-Justine, Montr´eal, Quebec, Canada; 2D´epartement de P´ediatrie, Universit´e de Montr´eal, Montr´eal, Quebec, Canada; 3McGill University and Genome Quebec Innovation Center, Montreal, Quebec, Canada Communicated by Richard Cotton It is likely that evolutionary differences among species are driven by sequence changes in regulatory regions. Likewise, polymorphisms in the promoter regions may be responsible for interindividual differences at the level of populations. We present an unbiased survey of genetic variation in 2-kb segments upstream of the transcription start sites of 28 protein-coding genes, characterized in five population groups of different geographic origin. On average, we found 9.1 polymorphisms and 8.8 haplotypes per segment with corresponding nucleotide and haplotype diversities of 0.082% and 58%, respectively. We characterized these segments through different summary statistics, Hardy-Weinberg equilibria fixation index (Fst) estimates, and neutrality tests, as well as by analyzing the distributions of haplotype allelic classes, introduced here to assess the departure from neutrality and examined by coalescent simulations under a simple population model, assuming recombinations or different demography. Our results suggest that genetic diversity in some of these regions could have been shaped by purifying selection and driven by adaptive changes in the other, thus explaining the relatively large variance in the corresponding genetic diversity indices loci. However, some of these effects could be also due to linkage with surrounding sequences, and the neutralists’ explanations cannot be ruled out given uncertainty in the underlying demographic histories and the possibility of random effects due to the small size of the studied segments. Hum Mutat 28(5), 441–450, 2007. r 2007 Wiley-Liss, Inc. KEY WORDS: DNA diversity; promoter regions; haplotypes; selective sweeps; human populations INTRODUCTION Patterns of DNA diversity in the human genome result from the stochastic nature of mutations and recombinations. They are further shaped by random genetic drift, by demographic history, and by natural selection. To understand the distinct contributions of the underlying genetic and evolutionary phenomena, we have to examine the genomic variation in individuals and populations from different geographic areas. So far, considerable effort has gone into the identification of polymorphisms which could be used as markers in linkage and association studies. Fewer studies have focused on DNA variability in particular genomic segments, and even fewer have investigated variation beyond two or three population groups. Likewise, much less attention has been given to DNA variants that could be classified as regulatory, in spite of their importance for understanding species evolution [King and Wilson, 1975], functional components of the genome [Carroll, 2005], and ultimately as phenotypic diversity and complex traits [Pastinen and Hudson, 2004]. On the other hand, there is an inherent difficulty in defining DNA segments and their variants that can be qualified as regulatory—in contrast, for example, to easily qualifiable alterations in the protein-coding sequences. In order to describe genetic diversity in cis-regulatory regions and, in particular, DNA variation involved in the control of transcription of the protein-coding genes, we used an operational definition of the promoter region as a 2-kb segment upstream of the transcription start site [Kim et al., 2005]. Previously, we described r 2007 WILEY-LISS, INC. DNA polymorphisms in 197 such regions surveyed for the presence of variants in a sample of 40 individuals representing five population groups distributed worldwide [Sinnett et al., 2006]. These loci were primarily chosen as candidate genes for their possible involvement in cancer [Belanger et al., 2005], drug response, inflammation, and/or displaying allelic imbalance [Pastinen et al., 2004]. In this article, we present a detailed characterization of genetic diversity in upstream regions of a subset of 28 of these genes, and we describe the corresponding allelic and haplotypic frequencies as well as their geographic distributions. The resulting diversity patterns portray different landscapes of genomic variability, suggesting as well that natural selection could have influenced the evolution of some of these segments. The Supplementary Material referred to in this article can be accessed at http://www.interscience.wiley.com/jpages/1059 -7794/suppmat. Received 9 March 2006; accepted revised manuscript 9 November 2006. Correspondence to: Damian Labuda, Centre de recherche, H oŒpital Sainte-Justine, 3175 C oŒte Sainte-Catherine, Montreal (Quebec) H3T 1C5, Canada. E-mail: damian.labuda@umontreal.ca Grant sponsor: Genome Quebec; Grant sponsor: Genome Canada; Grant sponsor: Canadian Institutes of Health Research (CIHR); Grant number: MOP67150; Grant sponsor: Rh oŒne-Alps Foundation. DOI 10.1002/humu.20463 Published online 1 February 2007 in Wiley InterScience (www. interscience.wiley.com).