Short Telomere Length as a Risk Factor for Myelodysplastic Syndromes: A Case-Control Study

BACKGROUND: Predisposition to myelodysplastic syndromes (MDS) may arise from accelerated cellular senescence of bone marrow stem cells, a hallmark of the aging process. Proliferative senescence can by triggered by critically shortened telomeres, and previous studies have reported shorter telomere length in myeloid cells of MDS patients as compared to healthy controls. Because myeloid cells are progeny of the malignant clone, it is unclear whether telomere length plays a pathogenetic role in the development of MDS or represents soley a disease specific phenotype. To investigate the role of telomere length in MDS susceptibility, we conducted a case-control study of 65 newly diagnosed MDS patients and 63 controls.

METHODS: Cases of MDS were recruited at the Moffitt Cancer Center (n=58) and through a study advertisement posted on the Myelodysplastic Syndromes Foundation website (n=7). MDS patients were eligible to participate if they were age 18 or older, had received no MDS treatment other than growth factors, and had no prior history of cancer. All MDS diagnoses were histologically confirmed and characterized by WHO and IPSS category. Controls were visitors who accompanied patients and were frequency-matched to MDS cases based on sex and age within 10 years. Eligible controls were not biologically related to the cases and did not have a history of cancer. All study participants completed a risk factor questionnaire, which included history of occupational exposures. DNA was extracted from heparinized whole blood samples, and telomere lengths were measured using real-time PCR. The ratio of telomere repeat copy number (T) to the copy number of a single-copy gene (beta-actin) (S) was determined using standard curves, where the T/S ratio is proportional to the cell’s average telomere length. In a subset of participants (n=18 cases and 15 controls), telomere length was also measured in CD15+ and CD19+ cells by quantitative fluorescence in situ hybridization assays using flow cytometry (flow-FISH). Peptide nucleic acid (PNA)-telomere probe binding in a population of cells with long telomeres (1301 cells) was used as both a reference internal standard in each flow-FISH assay and for quantification. In addition, a polymorphism in the telomerase reverse transcriptase gene (hTERT) previously shown to be associated with aplastic anemia (A1062T) was investigated using a Taqman allele differentiation assay. Telomere lengths were compared between MDS cases and controls using logistic regression with adjustment for age. To investigate potential determinants of telomere length among the MDS cases, analysis of covariance with age adjustment was used to compare telomere lengths by hTERT genotype and past occupational exposures.

RESULTS: Telomere lengths in whole blood were significantly shorter among MDS cases [mean (SD) T/S ratio for cases= 3.9 (2.3))] as compared to controls [mean (SD)=5.6 (4.6)] (p=0.01). Among MDS cases, telomere lengths in whole blood differed by MDS subtype, ranging from a mean T/S ratio of 2.2 in patients with refractory anemia [n=7] to 4.3 in patients with refractory anemia with ringed sideroblasts [n=9] (p=0.13). The A1062T polymorphism was present in 6% of cases versus 3% of controls (p=0.68) and was not associated with telomere length in either cases or controls. MDS cases were more likely than controls to report past occupational exposures to paints (p=0.11), insecticides (p=0.06) and organophosphates (p=0.06), and telomere lengths were shorter among those exposed to these substances than those who were not (p=0.10). Flow-FISH analysis showed that MDS cases had shorter telomere lengths in both CD15+ neutrophils (mean (SD) fluorescence units relative to 1301 = 10.0 (1.2) for cases and 11.5 (1.4) for controls; p=0.009) and CD19+ lymphocytes (cases = 11.7 (1.7), controls= 12.6 (1.4); p=0.15). Among MDS cases, telomere lengths in CD15+ and CD19+ cells were significantly correlated (Spearman correlation coefficient = 0.50, p=0.03).

CONCLUSION: Our findings of reduced age-adjusted telomere length in both neutrophils and B-cells of MDS suggests that accelerated telomere shortening may be a marker of genetic susceptibility to MDS.