Abstract
Abstract 2104
The phenotype of sickle cell disease is caused by sickle vasoocclusion and hemolytic anemia. Hemolysis in sickle cell anemia has been associated with complications that were presumed to result in part from vascular nitric oxide depletion due to scavenging by free plasma hemoglobin. Though plasma hemoglobin is a specific marker of intravascular hemolysis and red cell survival studies are the most definitive method of establishing the extent of hemolysis, these tests are rarely done and not available in large cohorts. However, the intensity of hemolysis can be estimated by the reticulocyte count, lactate dehydrogenase (LDH), aspartate aminotransaminase (AST) and bilirubin levels, all of which are commonly measured in cohort studies, although none of which is specific for hemolysis. We previously reported the results of a genome-wide association study (GWAS) of hemolysis where we used as a phenotype a new measure of the rate of intravascular hemolysis appropriate for cohort studies and GWAS. Using a principal component analysis of the commonly measured markers of hemolysis we derived a hemolytic score and found that the top SNPs associated with this score included a variant located in the first intron of NPRL3 (rs7203560; chr16p13.3, p=6.04×10−07) This result was replicated in two additional cohorts of 549 and 296 patients. We also established that while rs7203560 was associated with the ∝3.7 thalassemia gene deletion, when adjusted for HbF and ∝ thalassemia the association of NPRL3 with the hemolytic score remained significant (p=0.00375) and this association was also significant when examining only cases without ∝ thalassemia (p=0.02463). To further validate these results we studied 213 additional adult sickle cell anemia patients from King's College Hospital, London, UK. The mean age of these patients was 33 years. None had been treated with hydroxyurea and lab parameters obtained 3 months after, if transfused. Patients had similar clinical characteristics. The hemolytic score was calculated by using principal component analysis of the same markers of hemolysis. The SNPs associated with the hemolytic score in the primary study were genotyped in this cohort using TaqMan SNP genotyping assays according to standard Applied Biosystems protocol and their association with the derived hemolytic score studies using the same additive genetic model. The SNP rs7203560 replicated the association with hemolytic score (p= 0.03674) in this cohort. To examine the linkage disequilibrium (LD) structure of the region, we looked for conserved sequences in the α- globin cluster in multiple divergent species using the Basic Local Alignment Sequencing Tool (BLAST) to identify the approximate locations of the hypersensitive sites that are the major α-globin gene regulatory elements. On examination of the LD structure of SNPs in these regions with rs7203560, we found that rs7203560 was in LD with several SNPs located in and near the hypersensitive sites including rs2238368 (D'=1), rs2541612 (D'=0.89) and rs3331107 (D'=0.61). We hypothesize that rs7203560 is a marker for one or more variants in the major α-globin gene regulatory elements that down-regulate α-globin gene expression and cause a mild α thalassemia-like effect. In sickle cell anemia, perhaps by independently down-regulating expression of the α-globin genes, variants of the major ∝-globin gene regulatory loci reduce HbS concentration, lessen the polymerization potential of deoxy sickle hemoglobin and therefore retard hemolysis.
Disclosures:
No relevant conflicts of interest to declare.
Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer