Genetic variants that influence fetal hemoglobin expression from hydroxyurea treatment

Anu Marahatta, Jonathan Flanagan, Thad Howard, Nicole Mortier, William Schultz, Kathryn McElhinney, Leon Tshilolo, Thomas Williams, Peter Olupot-Olupot, Robert Opoka

Research output: Contribution to journalArticle

Abstract

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

Results: In the discovery GWAS step, no variant passed genome wide significance (p

Conclusions: This large GWAS using global cohorts of children with SCA and robust prospective HbF phenotype data has identified genetic predictors of HbF hydroxyurea treatment responses. Three novel genetic loci, PTPRD, RPH3AL, and ELL2 have SNVs associated with lower HbF responses. PTPRD is a protein tyrosine phosphatase receptor involved in cellular processes such as cell growth and differentiation, while RPH3AL, a rabphilin 3A like protein, is known to be involved in calcium-ion-dependent exocytosis. ELL2 is an elongation factor for RNA polymerase II and could modify RNA processing under the cytostatic effects of hydroxyurea. These genes and variants will be investigated to determine how they impact individual HbF responses to hydroxyurea treatment.

Original languageUndefined/Unknown
JournalPaediatrics and Child Health, East Africa
Publication statusPublished - 1 Nov 2020

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