Genetic Predictors of Hemoglobin F Response to Hydroxyurea in Sickle Cell Anemia

Abstract 241 Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia exhibit wide variability in their laboratory and clinical phenotype, suggesting additional genetic modifiers exist beyond the sickle mutation. One of the most powerful and reproducible disease modifiers is fetal hemoglobin (HbF) level. The most widely used and safest method for increasing innate fetal hemoglobin levels in patients with sickle cell anemia is hydroxyurea. While hydroxyurea has disease modulating effects outside of HbF induction, the majority of its benefit is directly related to the %HbF produced in response to the drug. Unfortunately, the amount of the HbF produced in response to hydroxyurea is highly variable between individuals, with induced HbF levels ranging from 5 to >30% even for compliant patients on similar dosing regimens at the maximum tolerated dose (MTD). Hydroxyurea is an ideal target for pharmacogenomics investigation, since there is strong concordance of HbF response to hydroxyurea within sibling pairs, and amount of HbF produced at MTD is a quantifiable and objective phenotype. To address the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated pediatric patients treated prospectively with hydroxyurea. We analyzed patients enrolled in the HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980) studies (n=183); all patients received an identical dose escalation regimen to MTD and had the most reliable HbF phenotypes available. To best identify genetic modifiers of hydroxyurea induction of HbF, we categorized study subjects according to their HbF response. Of a total cohort of 183 treated subjects, we identified 55 pediatric patients who represented the extreme ends of HbF response to hydroxyurea: 30 high responders (final HbF >30% and >25% change from %HbF at baseline) and 25 low responders (final HbF <20%, and <15% change from %HbF at baseline). There was no significant difference between high and low responders by age. Baseline fetal hemoglobin was similar between the two groups, suggesting that genetic predictors of drug response will differ from genetic regulators of endogenous HbF production. Absolute neutrophil count and hydroxyurea dose at MTD did not differ between high and low responders, evidence of uniform treatment. We performed whole exome sequencing on these 55 subjects and achieved 20X coverage in 95% of exonic sequences. In our statistical analysis, we compared nucleotide polymorphisms between low responders and high responders. Univariate testing identified ten nonsynonymous polymorphisms with p-values below 1×10−3, six of which are shown below. Additional candidate mutations with higher p-values were selected for further analysis based on known function in hematopoiesis or cell cycle arrest (Table). Whole exome sequencing genotyping was verified by TaqMan PCR or Sanger sequencing. Together, these variants represent excellent candidate genetic mutations to explain differences in HbF responses. These data represent the first examples of genetic predictors of HbF response to hydroxyurea using whole exome sequencing. Gene db SNP ID Amino acid change Function Direction of HbF response P-value PPP1R15A rs11541192 Gly312Ser Cell stress recovery Higher 1.87 × 10−4 HSD17B4 rs28943594 Met710Val Fatty acid oxidation Lower 4.16 × 10−4 HSD17B4 rs28943589 Lys122Asn Fatty acid oxidation Lower 4.18 × 10−4 FLVCR1 rs11120047 Ala52Pro Erythroid maturation Higher 4.21 × 10−4 LAMA5 rs6143021 His2036Arg Glomerular filtration Higher 4.21 × 10−4 ATP4A rs2733743 Val265Ala Iron absorption Higher 4.23 × 10−4 PPP1R15A rs611251 Val199Ala Cell stress recovery Higher 1.05 × 10−3 AKAP12 rs10872670 Lys19Glu Cell senescence Lower 1.28 × 10−3 SLC17A4 rs11754288 Ala372Thr Anion transporter Lower 1.86 × 10−3 RREB1 rs115093903 Leu983Ser Erythroid maturation Lower 2.31 × 10−3 DCHS2 rs61746132 Pro2676Leu Unknown Higher 2.90 × 10−3 SALL2 rs61743453 Pro840Arg Transcription factor Higher 9.17 × 10− Disclosures: Off Label Use: Hydroxyurea is FDA approved for adults but not children with sickle cell anemia.