Mutations in the Telomerase Reverse Transcriptase Gene Predisposes to Myelodysplastic Syndromes.

Abstract 415

Loss-of-function mutations in the telomerase complex genes, the enzyme responsible for maintaining the length of telomeres, are etiologic in patients with dyskeratosis congenita, an inherited bone marrow failure syndrome associated with mucocutaneous abnormalities, pulmonary fibrosis, esophageal stricture, liver cirrhosis, and an increased susceptibility to cancer, especially acute myeloid leukemia and squamous cell carcinoma. Heterozygous mutations abrogate telomerase's enzymatic ability to elongate telomeres by a mechanism of haploinsufficiency. Patients show accelerated telomere shortening in leukocytes, which eventually cause cellular senescence and apoptosis of hematopoietic precursors. Critically short telomeres lose their ability to sustain chromosomal stability, promoting end-to-end fusions and breakage-fusion-bridge cycles. Constitutional telomerase mutations also are found in patients with apparently acquired aplastic anemia, pulmonary fibrosis, and acute myeloid leukemia (AML). In AML, telomerase mutations correlate with cytogenetic abnormalities, especially trisomy 8 and inversion 16, and it appears that dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia cells by selection of stem cells with defective DNA damage responses that are prone to chromosomal instability. In the present study, we investigated whether mutations in genes for components of telomerase also occur in patients with myelodysplastic syndromes (MDS), and correlated mutation status with chromosomal changes. We screened DNA extracted from blood or bone marrow cells from 359 patients diagnosed with MDS and 528 healthy individuals for sequence variations in the telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) genes. Fourteen patients (4%) were heterozygous for telomerase gene variants in comparison to 7 healthy controls (1.3%; P=0.02, Fisher's exact test). Three mutant patients had refractory anemia; three had secondary AML; three had refractory anemia with ringed sideroblasts; two had chronic myelomonocytic leukemia; one had refractory anemia with excess blasts-2; one had refractory cytopenia with multilineage dysplasia; and one had MDS-unclassified. Vectors containing the TERT gene were mutagenized with novel gene variants and introduced into VA13 cells along with the TERC gene (VA13 cells are deficient for telomerase expression and maintain their telomeres via asymmetric telomeric recombination). Cell lysates from cells transfected with MDS-associated TERT showed reduced telomerase enzymatic activity in comparison to wild-type TERT. Using conventional karyotyping, six mutant patients had abnormal cytogenetics, and trisomy 8 was the most common cytogenetic abnormality observed in telomerase-mutant patients (three cases). Single nucleotide polymorphism array (SNP-array) analysis was performed in twelve telomerase-mutant patients (6.0 array in nine, and 250K array in three patients). Eleven of the twelve patients analyzed showed large lesions or copy number variations that have not been previously reported in normal individuals. More common were large losses in the long arm of the acrocentric chromosome 13, chromosome 7, and gains in the long arm of chromosome 3 and trisomy 8. One patient also showed somatic uniparental dissomy. Our results indicate that hypomorphic telomerase mutations may predispose to the development myelodysplastic syndromes. Myelodysplastic cells of patients carrying a telomerase mutation showed significant number of chromosomal abnormalities either by conventional cytogenetics or SNP-array in a proportion higher than that usually observed for MDS, suggesting increased chromosomal instability. In conclusion, short and dysfunctional telomeres in hematopoietic progenitors reduce telomere protection, compromising chromosomal stability and integrity, and our results indicate that these genomic changes may predispose for the selection of abnormal clones and the development of myelodysplastic syndromes.