Identification and Functional Characterization of Novel Telomerase Variant Alleles in Patients with Bone-Marrow Failure Syndromes

As multiple studies have shown a link between mutations in telomerase components and the disease pathogenesis of bone-marrow failure syndromes (BMFS), we screened blood or marrow cells from patients with various forms of blood disorders for novel pathogenic mutations in the telomerase hTERC and hTERT genes. We identified several heterozygous mutations in these genes, which were observed only in patient samples and not in controls. As these mutations are predicted to affect proper telomerase function, we introduced these and other mutations identified previously in patients with other forms of blood disorders into telomerase-negative cells in order to reconstitute telomerase enzymatic activity and to assess the effect of the individual mutations on modulating a wild-type telomerase function. All disease-associated mutations disrupted telomerase function to various degrees and most modulated a wildtype telomerase enzymatic function by haploinsufficiency based on results obtained from the telomeric repeat amplification protocol (TRAP). We also tested several previously reported natural sequence changes in the promoter region of the hTERC gene for their effect on gene expression and telomerase function via luciferase-reporter assay, TRAP, and Northern blot. The results obtained from these studies appear to contradict those that have recently been reported for some of the naturally occurring hTERC-promoter associated mutations. This study, therefore, offers new insights into the mechanism of natural telomerase mutations in regulating telomere lengths and marrow stem cell renewal capacity in patients with hematological disorders.