ALBERT

Description: Introduction: Hydroxyurea is a potent therapeutic agent for sickle cell anemia (SCA), and treatment at maximum tolerated dose (MTD) is becoming the standard of care. Hydroxyurea exerts its disease-modifying effects primarily through induction of fetal hemoglobin (HbF), although the cellular and molecular mechanisms by which hydroxyurea increases HbF expression remain unclear. Children with SCA treated with hydroxyurea at MTD have substantial phenotypic variation, however, as some have higher HbF responses than others. We hypothesized that unknown quantitative trait loci modulate the pharmacological induction of HbF, so we performed a large genome wide association study (GWAS) of hydroxyurea-associated HbF responses for children with SCA treated prospectively with dose escalation to MTD. Methods: We analyzed genomic DNA from 831 children with SCA enrolled in pediatric research trials from the US (HUSTLE, SWiTCH, TWiTCH), the Caribbean (EXTEND, SACRED) and sub-Saharan Africa (REACH, NOHARM); all of these trials reported robust treatment responses with average HbF 〉20%. Study participants received hydroxyurea with dose escalation to MTD based on mild myelosuppression. Whole blood DNA was genotyped using the H3Africa SNP array (Illumina) with whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform. A transformed z-score for each study cohort gave a standardized measure of HbF induction relative to their steady-state level and their treatment HbF level at MTD. These standardized z-score HbF values were then used as a continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, using age, hydroxyurea dose at MTD, and sex as co-variates. We first performed an initial GWAS discovery using hydroxyurea response data from four distinct African populations (n=377). Single nucleotide variants (SNVs) with nominal significance (p