Telomerase, hTERT and splice variants in patients with myelodysplastic syndromes

Highlights

• The telomere length in PBMCs of patients with MDS cases was significantly shorter compared to controls (n = 30, p = 0.002).

• MDS patients had significantly higher basal telomerase activity (p = 0.022) and higher total hTERT (p = 0.007), α+β+ hTERT variant (p = 0.016) and α+β− hTERT variant expression than control.

• The ratio of α+β− transcript to α+β+ transcript was significantly increased in cases (p = 0.039).

Abstract

Telomeres are specialized structures maintaining chromosome integrity during cellular division and preventing from premature senescence and apoptosis. The rate-limiting component of telomerase is human telomerase reverse transcriptase (hTERT), for which multiple transcripts exist. The aim of this work was to characterize hTERT splice variants in MDS and its relation to telomerase activity, telomere length and hTERT expression. The telomere length in PBMCs of patients with MDS cases was significantly shorter compared to controls (n = 30, p = 0.002). MDS patients had significantly higher basal telomerase activity (p = 0.022) and higher total hTERT (p = 0.007), α+β+ hTERT variant (p = 0.016) and α+β− hTERT variant expression than control. The ratio of α+β− transcript to α+β+ transcript was significantly increased in cases (p = 0.039). This study provided a detailed insight into the hTERT transcript pattern in MDS while correlation analysis showed that only telomerase activity was significantly correlated with total hTERT expression in MDS.

Introduction

Mydysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias and potential to acute myeloid leukemia (AML) progression in approximately 30% of cases [1], [2]. The early manifestations of MDS are relatively well conserved and include inflammation within the bone marrow microenvironment coupled to apoptosis and excessive proliferation [3], [4], [5]. Evidence in MDS displayed accelerated telomere shortening within the myeloid progenitor and stromal cells [6], [7], [8], [9], indicates that a defect occurs within the hematopoietic compartment [10].

Telomeres are a repetitive hexanucleotide (TTAGGG) region providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis [11]. Telomerase is a ribonucleoprotein complex composed of two essential components: a catalytic subunit with reverse transcriptase activity (human telomerase reverse transcriptase [hTERT]) and an RNA subunit human telomerase RNA (hTR). Telomerase expression exists in normal germ cells and other stem cells, which have self-renewal and high pro-liferative potential. Its absence from most normal somatic cells is believed to contribute to eventual senescene and limited cellular life span [12], whereas its reactivation in immortalized cells has been associated with the unlimited growth potential required for malignancy [13]. hTERT is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Accordingly, expression of hTERT was also found to correlate with poor clinical outcome in hematologic malignancies [6].

Up to now, at least six splice variants of hTERT have been described [14]. The most widely studied variants involving in splicing located at two main sites: the α splice site produces a 36-bp in frame deletion within the conserved reverse transcript motif A and its splice variant was found to be a dominant negative inhibitor of telomerase activity; and the splicing at β site can cause a 183-bp deletion and non-sense mutation that truncates the protein, and result in a non-active TERT protein and catalytically inactive telomerase. Only the full-length (FL) transcript (α+/β+) containing the A and B reverse transcriptase motifs, translates into a functional protein. Splicing at either site can occur independently or in combination to produce three variants from the full-length α+/β+: α−/β+, α+/β−, and α−/β− [14]. The importance of these reverse transcriptase splice variants is highlighted during early human fetal development [15] and embryo maturation [16]. It is unclear whether spliced hTERT is important in determining telomerase activity and telomere length. Knowledge of the different hTERT mRNA splice variants is an important issue to evaluate telomerase expression. Few studies have directly examined the differences of hTERT splice variants in PBMCs between MDS and healthy controls.

In the current study, we examined hTERT expression and telomerase activity in 30-MDS patients, and detected the four isoforms (full length, α− deletion, β− deletion and α−β− deletion) by Taqman-based quantitative real-time reverse transcription (qRT)-PCR and their correlation with telomerase activity and telomere length. The aim of this study is going to characterize the role of hTERT mRNA splicing in the regulation of this enzyme and telomere length in MDS patients.