Identifying sickle cell anemia modifying genes by exome analysis

Abstract

Sickle cell anemia (SCA) is one of the most common monogenic disorders worldwide. About 3,000 newborns are affected with SCA each year in Brazil and it is the most common genetic disease in France and France overseas territories (international collaboration in this project). SCA is characterized by a very heterogeneous clinical course, ranging from mild to life-threatening conditions. This high inter-individual variability remains unexplained. While necessary, HbS alone cannot account for this heterogeneity. Persistence of fetal hemoglobin (HbF) and concomitant ±-thalassemia are the best-characterized SCA modifiers, but do not explain by themselves the extent of the phenotypic variability. Altogether, these observations have led to the hypothesis of a multifactorial control of SCA complications, involving both genetic and environmental factors. Initially, the quest of modifier genes has been undertaken via a candidate gene approach with a mitigated success. Actually, the only convincing association described so far using this type of genotype-phenotype association study, is that of a functional polymorphism in the UGT1A gene and cholelithiasis; In contrast, none of the many different gene associations reported with different complications have been replicated despite specific attempts for some of them.Aim: our aim is to use exome analysis to identify modifier genes involved in the occurence of a major SCA complication occurring during childhood, i.e. acute splenic sequestration (ASS). This complication has been choosen because of: i. Its occurrence early in life, ii. Its consensual definition, iii. Its frequency and Its significant impact on SCA morbidity and mortalityPatients and Research protocol: SCA homozygous patients (SS) identified via newborn screening currently older than 6yoa and regularly followed at the following Sickle Cell Centers are eligible for this study will be including during the annual follow-up visit. The Sickle Cell Unit at the Hemocentro, University of São Paulo Medical School, (Ribeirão Preto, Brazil), The SCD Reference Center of French West-Indies (Guadeloupe, Martinique) and Guiana, with a coverage higher than 97% for both. The SCD Reference Center of the Greater Paris (Ile de France), that using a targeted approach (mainland France).We will perform a two-steps strategy. The identification of modifier genes will be undertaken by exome analysis of 15 discordant pairs (SCA siblings older than 6 year old age (yoa), one with a past history of recurrent ASS and one with no history of ASS). The association between these two complications and the selected variants will be confirmed by a targeted analysis in 600 unrelated cases and 600 controls.Experimental scheme: DNA will be purified by conventional procedures. Phenotypic data pertinent to the study will be collected from the medical file of the patients.For the exome approach, intra-familial cases and controls will be selected. Such strategy, based on the analysis of discordant SCA sibling pairs (brothers or sisters) will reduce the rate of false positive results related to the recent admixture of our populations of interest and increase the statistical power of the analysis since brothers and sisters share 25% of their genome in average. Furthermore, only patients with recurrent ASS will be selected in order to increase the probability of detecting variants associated with the higher penetrance. All variants shared between discordant pairs will be discarded from further analysis. Filtering processes will be performed to retrieve variants potentially altering protein function, according to predictive tools, i.e. truncating, splice site variants and missense mutations that are predicted to alter protein function. Variants potentially altering gene expression will also be retrieved. Two mains statistical approaches will be used to analyze variants identified by exome analysis, namely collapsing and aggregation methods. (AU)