The role of telomerase and chromosome healing in patients with terminal deletions
thesisposted on 15.12.2014, 10:38 by Helen. Varley
A simple telomere-anchored PCR-based method was used to isolate telomere-junction clones from a number of individuals. Analysis of clones led to the isolation of novel chromosome ends, including a low level polymorphic telomere, present in 2.4 % of the Caucasian population. The sequence adjacent to this telomere shows homology to a subterminal repeat found at internal locations on 4p, 4q, 10q and 22q in most individuals. A number of modifications were made to the telomere-anchored PCR strategy, including generation of telomere variant repeat probes to detect many normal chromosome ends, and the addition of a filter hybridisation selection step to increase enrichment for telomere-repeat arrays from genomic DNA. The modified strategy was used successfully to isolate a candidate terminal deletion breakpoint from a patient with a deletion of 22q. The telomere-adjacent sequence does not show homology to subterminal repeats and is unique to chromosome 22. It shows 96.2 % sequence identity to a BAC clone mapped to 22ql3.3, consistent with initial RFLP data obtained by another group. The sequence is located adjacent to a telomere in the patient, but not in 87 unrelated individuals, or the patient's parents, indicating that it is a de novo telomere repeat array. The telomere appears to contain TTAGGG repeats, without variants. The lack of subterminal sequence at the breakpoint, and array of only TTAGGG repeats strongly suggests that the truncated chromosome was healed via direct addition of telomere repeats by the enzyme telomerase. Comparison of the sequence around this putative breakpoint region with sequence generated from a 7q32 breakpoint, and previously characterised breakpoints at 16pl3.3, did not reveal any sequence features common to these regions, apart from a high density of dispersed repeats. The lack of conserved sequence features suggests that telomerase does not have strict sequence requirements for healing broken chromosomes in vivo.