Balanced polymorphism in bottlenecked populations: The case of the CCR5 5= cis-regulatory region in Amazonian Amerindians Rodrigo F. Ramalho a, *, Eduardo J.M. Santos b , JoÄo F. Guerreiro b , Diogo Meyer a a Universidade de Sâo Paulo, Instituto de Biociéncias, Departamento de Genètica e Biologia Evolutiva, Sâo Paulo, SP, Brasil b Universidade Federal do Parà, Centro de Ciéncias Biològicas, Laboratòrio de Genètica Humana e Mèdica, Belèm, PA, Brasil ARTICLE INFO Article history: Received 5 January 2010 Accepted 26 May 2010 Available online 9 June 2010 Keywords: 5= cis-regulatory region of CCR5 gene Amerindians Balancing selection Demography Genetic drift ABSTRACT The 5= cis-regulatory region of the CCR5 gene exhibits a strong signature of balancing selection in several human populations. Here we analyze the polymorphism of this region in Amerindians from Amazonia, who have a complex demographic history, including recent bottlenecks that are known to reduce genetic vari- ability. Amerindians show high nucleotide diversity ( = 0.27%) and significantly positive Tajima’s D, and carry haplotypes associated with weak and strong gene expression. To evaluate whether these signatures of balancing selection could be explained by demography, we perform neutrality tests based on empiric and simulated data. The observed Tajima’s D was higher than that of other world populations; higher than that found for 18 noncoding regions of South Amerindians, and higher than 99.6% of simulated genealogies, which assume nonequilibrium conditions. Moreover, comparing Amerindians and Asians, the Fst for CCR5 cis- regulatory region was unusually low, in relation to neutral markers. These findings indicate that, despite their complex demographic history, South Amerindians carry a detectable signature of selection on the CCR5 cis-regulatory region.  2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. 1. Introduction The CC-chemokine 5 receptor (CCR5) binds the -chemokines CCL2, CCL3, CCL4, and CCL5, playing a fundamental role in the innate immune response and inflammatory processes [1]. It is ex- pressed not only in cells and organs involved in immunity, but also in cells of the central nervous system and on the epithelium and endothelium of other tissues [2]. In addition to its constitutive function, CCR5 is used by the human immunodeficiency virus (HIV) as a co-receptor when entering a cell [3]. This explains why indi- viduals homozygous for a 32-bp deletion (delta32) in the open reading frame (ORF) of the gene are relatively resistant to infection by HIV and heterozygous individuals show a delay in disease pro- gression [4]. Great variability in the expression of CCR5 has been observed in T cells from homozygous individuals for the delta32 wild-type allele [5]. However, the polymorphisms found in the coding region of the CCR5 gene could not explain the variation in disease risk among individuals and populations because they are, in most cases, found in low frequencies [6–8]. As an exception, Boldt et al. [9] described two new nonsynonymous mutations in Amerindians, one of them being relatively common (10%). These findings motivated the study of the promoter region of CCR5, revealing that specific haplotypes were associated with in- creased or decreased rates of disease progression in HIV-infected individuals [10]. The CCR5 gene has a complex structure, with two promoters and an alternatively spliced cis-regulatory region located in the 5= UTR of the primary transcript [11]. These features suggest that the 5= UTR plays an important role in pre- and post-transcriptional con- trol of CCR5 expression and therefore could be an important target for natural selection. Balancing selection upon the 5= UTR region of CCR5 is supported by several lines of evidence [12]: (a) its diversity is fourfold higher than that of other noncoding regions of the human genome [13]; (b) population differentiation, as measured by Fst, is significantly lower than for other markers; (c) there are more polymorphic sites at inter- mediate frequency than expected under neutral-equilibrium condi- tions, resulting in positive and significant Tajima’s D value; and (d) haplotypes are connected by a network exhibiting two main clus- ters, separated by a relatively high level of divergence. Bamshad et al. [12], showed that the high genetic diversity observed for CCR5 cis-regulatory region could not be accounted for by a high recombination or mutation rate, and that the low differentiation among populations was not an artifact of the sampling strategy used, strengthening the claim that this region is under balancing selection. Interestingly, several examples of balancing selection upon reg- ulatory regions of genes involved in immunity have recently been found, including HLA-G [14], HLA-DPA1 and DPB1 [15]. Specifically * Corresponding author. E-mail address: rfr@ib.usp.br (R.F. Ramalho). Human Immunology 71 (2010) 922–928 Contents lists available at ScienceDirect 0198-8859/10/$32.00 - see front matter  2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.05.022