Telomere Shortening and Genomic Instability: Primary Cells from Patients with Telomere Repair Complex Mutations Are Susceptible to End-to-End Chromosome Fusion and Aneuploidy.
Authors: Sloand Elaine M. , Calado Rodrigo , Shah Simant , Pfannes Loretta , Blancato Jan , Young Neal 
Published: October 2019

Abstract
Telomeres that cap the ends of chromosomes, protect them from recombination, end-to-end fusion, and recognition as damaged DNA. Telomere shorteningmay result in cell cycle arrest and senescence or in genomic instability that precedes malignancy. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex, mainly the telomerase reverse transcriptase (TERT) and its integral RNA template (TERC). Mutations in TERC and TERT cause progressive telomere shortening; short telomeres are found in constitutional marrow failure syndromes and also in apparently acquired aplastic anemia (Fogarty PF et al, Lancet362:1628, 2003;. Yamaguchi H et al, NEJM352:1413, 2005). Myelodysplasia and acute leukemia are often present in these kindreds and associated with pathogenic mutations. The mechanism resulting in genomic instability in TERT and TERC-deficient individuals is unclear. End-to-end chromosome fusion in TERT-”knock-out” embryonic mouse stem cells are seen after multiple passages in culture and are associated with profound telomere shortening and the development of aneuploidy (Lee HW et al. Nature 9:569–74, 1998). End-to-end fusion of sister chromatids presumably results in disruption of chromosome segregation during metaphase. In the present study, we examined blood and marrow cells from 7 patients with TERT, and TERC gene mutations; 10 healthy young controls; and 2 individuals older than age 60 years of age, as well as 6 umbilical cord bloods. Leukocytes with TERT or TERC mutations had short telomeres as previously measured by flow-fluorescent in situ hybridization (FISH) and confirmed by Southern hybridization (mean telomere shortening in comparison to age-matched controls, 3.4 ± 1.7 kb) and low telomerase enzymatic activity (144±13 vs. 270±56 respectively; P<0.02)compared to normal controls, using the telomeric repeat amplification protocol assay. Bone marrow mononuclear cells were cultured for several days, synchronized with colcemid, and metaphase spreads were prepared for FISH using a telomeric probe. Samples from 4 patients who had significant numbers of metaphases demonstrated end-to-end fusions of chromosomes in 2- -20 % of metaphases. One elderly healthy donor demonstrated small numbers of metaphases with end to end fusions; no fusions were identified in any cord blood samples or in the 5 samples from younger donors. To measure aneuploidy, bone marrow cells from 5 patients were cultured for two weeks with 400ng/mL of G-CSF. Three patients with TERT and two patients with TERC mutant cells showed trisomy 8 (4–24% of interphases, mean=12%), with fewer samples (N=2) showing monosomy 7 (2–40%, mean 14%) and trisomy 9 by FISH; none of the ten samples from young healthy controls showed any anueploidy after culture (p=0.007). We have established links between telomere shortening, end-to-end chromosome fusion, and genomic instability, predicted from murine models, in human cells. Telomere shortening-secondary to genetic lesions in crucial genes of the repair complex and perhaps also due to normal aging (and, in theory, marrow “stress”) may underlie the genomic instability that leads to aneuploidy, providing a mechanism for chromosome aberrations observed in patients with dyskeratosis congenita, in “clonal evolution” of aplastic anemia, and in the most frequent form of bone marrow failure in the elderly, myelodysplasia.