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

Abstract

Abstract 241Although 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.Genedb SNP IDAmino acid changeFunctionDirection of HbF responseP-valuePPP1R15Ars11541192Gly312SerCell stress recoveryHigher1.87 × 10−4HSD17B4rs28943594Met710ValFatty acid oxidationLower4.16 × 10−4HSD17B4rs28943589Lys122AsnFatty acid oxidationLower4.18 × 10−4FLVCR1rs11120047Ala52ProErythroid maturationHigher4.21 × 10−4LAMA5rs6143021His2036ArgGlomerular filtrationHigher4.21 × 10−4ATP4Ars2733743Val265AlaIron absorptionHigher4.23 × 10−4PPP1R15Ars611251Val199AlaCell stress recoveryHigher1.05 × 10−3AKAP12rs10872670Lys19GluCell senescenceLower1.28 × 10−3SLC17A4rs11754288Ala372ThrAnion transporterLower1.86 × 10−3RREB1rs115093903Leu983SerErythroid maturationLower2.31 × 10−3DCHS2rs61746132Pro2676LeuUnknownHigher2.90 × 10−3SALL2rs61743453Pro840ArgTranscription factorHigher9.17 × 10− Disclosures:Off Label Use: Hydroxyurea is FDA approved for adults but not children with sickle cell anemia.