Original Article Mechanistic Studies of the Negative Epistatic Malaria-protective Interaction Between Sickle Cell Trait and α + thalassemia D. Herbert Opi a,b , Lucy B. Ochola a , Metrine Tendwa a , Bethsheba R. Siddondo a , Harold Ocholla a , Harry Fanjo a , Ashfaq Ghumra b , David J.P. Ferguson c , J. Alexandra Rowe b,1 , Thomas N. Williams a,d, ⁎ ,1 a Kenya Medical Research Institute-Wellcome Trust Research Programme, PO BOX 230-80108 Kilifi, Kenya b Centre for Immunity, Infection and Evolution, Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, EH9 3FL, United Kingdom c Nuffield Department of Clinical Laboratory Science, University of Oxford, John Radcliffe Hospital, OX3 9DU Oxford, United Kingdom d Department of Medicine, Imperial College, St Mary's Hospital, Praed Street, London W21NY, United Kingdom abstract article info Article history: Received 12 September 2014 Received in revised form 7 October 2014 Accepted 9 October 2014 Available online 13 October 2014 Keywords: Malaria Sickle cell trait α + thalassemia Epistasis Cytoadhesion PfEMP1 Red blood cells Background: Individually, the red blood cell (RBC) polymorphisms sickle cell trait (HbAS) and α + thalassemia protect against severe Plasmodium falciparum malaria. It has been shown through epidemiological studies that the co-inheritance of both conditions results in a loss of the protection afforded by each, but the biological mech- anisms remain unknown. Methods: We used RBCs from N 300 donors of various HbAS and α + thalassemia genotype combinations to study the individual and combinatorial effects of these polymorphisms on a range of putative P. falciparum virulence phenotypes in-vitro, using four well-characterized P. falciparum laboratory strains. We studied cytoadhesion of parasitized RBCs (pRBCs) to the endothelial receptors CD36 and ICAM1, rosetting of pRBCs with uninfected RBCs, and pRBC surface expression of the parasite-derived adhesion molecule P. falciparum erythrocyte mem- brane protein-1 (PfEMP1). Findings: We confirmed previous reports that HbAS pRBCs show reduced cytoadhesion, rosetting and PfEMP1 expression levels compared to normal pRBC controls. Furthermore, we found that co-inheritance of HbAS with α + thalassemia consistently reversed these effects, such that pRBCs of mixed genotype showed levels of cytoadhesion, rosetting and PfEMP1 expression that were indistinguishable from those seen in normal pRBCs. However, pRBCs with α + thalassemia alone showed parasite strain-specific effects on adhesion, and no consistent reduction in PfEMP1 expression. Interpretation: Our data support the hypothesis that the negative epistasis between HbAS and α + thalassemia ob- served in epidemiological studies might be explained by host genotype-specific changes in the pRBC-adhesion prop- erties that contribute to parasite sequestration and disease pathogenesis in vivo. The mechanism by which α + thalassemia on its own protects against severe malaria remains unresolved. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 1. Introduction The burden of malaria, currently estimated at over 2 million epi- sodes of clinical disease and 655,000 deaths annually (WHO, 2012), has exerted strong selection on the human genome, leading to the oc- currence at high frequencies of a number of host-protective polymor- phisms (Kwiatkowski, 2005). Some of the best-documented examples are inherited disorders of hemoglobin that include sickle hemoglobin (HbS) and α + thalassemia (Williams, 2006). Carriers of HbS, which results from the substitution of valine for the usual glutamic acid at the 6th position of the β-globin amino acid chain, have sickle cell trait (HbAS), a clinically silent condition that is associated with a high degree of protection against all forms of clinical malaria (Taylor et al., 2012; Williams et al., 2005a). α + thalassemia, which results from a deletion of one of the paired α-globin genes on chromosome 16 (-α), has been shown to protect against severe and fatal malaria in both its het- erozygous (-α/αα) and homozygous (-α/-α) forms (Taylor et al., 2012; Williams et al., 2005b). The significant overlap in the geographic distributions of these two conditions means that they are frequently co- inherited (Flint et al., 1998). Nevertheless, rather than conferring an ad- ditive advantage, co-inheritance of both HbAS and α + thalassemia is as- sociated with the loss of the malaria-protection that is afforded by each polymorphism individually (Williams et al., 2005c; Crompton et al., 2008; May et al., 2007), through an unknown mechanism. EBioMedicine 1 (2014) 29–36 ⁎ Corresponding author at: KEMRI/Wellcome Trust Programme, Centre for Geographic Medicine Research Coast, P.O. Box 230-80108, Kilifi, Kenya. E-mail address: tom.williams@imperial.ac.uk (T.N. Williams). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ebiom.2014.10.006 2352-3964/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com i An update to this article is included at the end