Leukocyte Telomere Length Correlates with Disease Severity and Inflammation in Sickle Cell Disease

Telomeres are specialized nucleoprotein structures that protect chromosome ends. The loss of telomeric structure permits DNA-damage and results in cellular senescence and apoptosis. Short telomeres may be due to either impaired telomere elongation due to telomerase defect as observed in dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis or caused by "stress" hematopoiesis and increased cell proliferation due to chronic inflammation and oxidative stress. As bone marrow demand and inflammation are characteristics of sickle cell disease (SCD), we sought to evaluate telomere length in SCD patients. Mean telomere length was measured in peripheral blood leukocytes by quantitative polymerase chain reaction (qPCR), as we have previously described (Gutierrez-Rodrigues, PLoS ONE 2014; 9(11):e113747). The telomere length for each sample was determined using the telomere to single copy gene ratio (T/S ratio) with the calculation of the DCt [Ct^(telomere)/Ct^{(single gene)} ]. To validate the qPCR results, we performed terminal restriction fragment (TRF) analysis for 17 samples with long and short telomeres, according to the manufacturer's instructions (TeloTAGGG Telomere Length Assay - Roche Applied Science, Mannheim, Germany). Tumor necrosis factor-alpha (TNF-α) and interleukin 8 (IL-8) were assessed as pro-inflammatory markers, measured in serum samples, in duplicate, using ultra sensitive ELISA kits. Spearman's rank correlation coefficient was used to analyze bivariate associations between telomere lengths, hemolysis markers and inflammation markers. We compared telomere length according to the diagnosis, use of hydroxyurea and gender using Wilcoxon rank sum test. All p values ≤0.5 were considered significant. In all of the analysis we used age-ajusted T/S ratios. A total of 91 adult SCD patients were included in the study, 51 with HbSS, 38 with HbSC and 2 with HbSβ. Fifty-one were off hydroxyurea and 40 were taking the drug, with a median dose of 1g/day (range 500-1750mg/day). We compared age-adjusted T/S ratio of SCD patients to 261 healthy controls and found significantly shorter telomeres in SCD patients (p<0.0001). When we compared HbSS patients to HbSC and HbSβ patients HbSS patients had significantly shorter telomeres (p<0.0001). Telomere length also was shorter in patients on hydroxyurea when compared to those off the drug (p=0.02). We found no difference in T/S ratio according to gender or age in the SCD cohort. To evaluate whether inflammation or hemolysis were associated with telomere attrition in SCD patients, we analyzed the associations between T/S ratios and hemolysis (hemoglobin, hematocrit, lactate dehydrogenase, indirect bilirubin, reticulocyte counts) and inflammation markers (IL-8, TNF-α, total leucocyte, neutrophil, lymphocyte and monocyte counts). We found a significant positive correlation between hemoglobin level and T/S ratio (r=0.29; p=0.004), and a negative correlation between lymphocyte counts and telomere length (r=-0.29; p=0.005), and between IL-8 serum levels and telomere length (r=-0.4; P=0.02). SCD patients presented shorter telomeres in comparison to age-matched health controls. Telomere erosion in SCD appeared to correlate with disease severity and to treatment with hydroxyurea. Our results also indicate that telomere attrition correlated with the intensity of anemia and inflammatory markers.