Genetic Association of Clinical Complications for Genes Differentially Expressed in Reticulocytes of Sickle Cell Anemia
Authors: Zhang Xu , Shah Binal N. , Hassan Taif , Nekhai Sergei , Machado Roberto , Gordeuk Victor R. 
Published: February 2021

Introduction
Sickle cell anemia (SCA) is characterized by hemolysis, a tissue hypoxia-driven increase in erythropoietin production, and a marked reticulocytosis reflecting stress erythropoiesis. Reticulocytes in SCA constitute 4-15% or more of peripheral red blood cells. Sickle reticulocytes express adhesion molecules that are normally cleared by exosome shedding, including α4β1 integrin, which is thought to contribute to vaso-occlusive crises by binding to vascular endothelial cell vascular cell adhesion molecule 1. Our recent study of the reticulocyte transcriptome in SCA versus healthy African Americans, using RNA-Seq, revealed differential genes that were implicated in a broad range of biological processes including terminal erythroid differentiation, the unfolded protein response, and altered membrane systems. In this study, we assessed the genetic association of these differential genes with disease complications relevant to sickle reticulocytes.
Methods
We analyzed vaso-occlusive complications including episodic severe pain and chronic avascular necrosis. We also analyzed hemolysis and anemia measured by a hemolysis index and hemoglobin concentration, respectively. The patient cohort combined 525 adolescent and adult patients from Walk PHaSST and 377 pediatric patients from PUSH. Genomic DNA isolated from peripheral blood mononuclear cells of Walk-PHaSST and PUSH patients were genotyped using Illumina Human 610-Quad SNP array and the genotypes were phased and imputed to 1000 genomes phase 3 data. Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) &gt;0.01 and imputation r2&gt;0.7 were tested for gene-wise genetic association using SKAT. For severe pain and avascular necrosis, the covariates included age, gender, β globin genotype severity (HHbSS and HbSβ0-thalassemia versus HbSC and HbSβ+-thalassemia), and population stratification. For hemolysis and hemoglobin concentration, the covariates included age, gender, β globin genotype severity, alpha thalassemia, hydroxyurea treatment, recent blood transfusion, and population stratification.
Results
A total of 111 genes were analyzed. These genes were classified into 13 functional categories based on published research (transcription, post-transcription, translation, histone modification, cell cycle arrest, mitophagy, metabolism, iron homeostasis, antioxidative system, unfolded protein response, membrane skeleton and structural proteins, membrane adhesion molecules and blood group antigens, and membrane transporters). For each functional class, the number of significant gene-wise associations the functional class contains was compared to the number of significant associations from the other classes combined. Defining significance of gene-wise association at nominal P=0.05, we found that the "Unfolded protein response" contained more significant gene-wise associations with severe pain than the other functional classes (one-sided Fisher's exact test P=0.046), driven by genes FUCA1 (alpha-L- fucosidase) and CLN8 (associated with endoplasmic reticulum function). "Membrane skeleton and structural proteins" showed a trend of enrichment for hemolysis (P=0.057), driven by genes MYO5B and CD2AP, and for avascular necrosis (P=0.075), driven by genes MYLK, CAVIN2 and PALLD.
Defining the significance of gene-wise association at a less stringent nominal P=0.1, "membrane adhesion molecules and blood group antigens" showed a trend of enrichment in association with severe pain (P=0.097), driven by genes GYPA, ITGA4 and ITGB1, the latter two encoding α4β1 integrin subunits which are abnormally present on the reticulocyte membrane and have been implicated in sickle vaso-occlusion. Regional scans revealed the SNPs with the most significant associations within these genes.
Discussion
Our results reveal enrichment of genetic association with vaso-occlusive and hemolytic complications in gene functional categories that showed altered expression in sickle reticulocytes. Future study is needed to validate these associations.

Disclosures
Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding; Ironwood: Research Funding; Novartis: Consultancy.