Germline mutations in Protection of Telomeres 1 in two families with Hodgkin lymphoma

Abstract

In a previous whole exome sequencing of patients from 41 families with Hodgkin lymphoma, we identified two families with distinct heterozygous rare coding variants in POT1 (D224N and Y36H), both in a highly conserved region of the gene. POT1 D224N mutant did not bind to a single-stranded telomere oligonucleotide in vitro suggesting the mutation perturbs POT1's ability to bind to the telomeric G-rich overhang. Human HT1080 cells expressing POT1 D224N and lymphoblastoid cells carrying Y36H both showed increased telomere length and fragility in comparison to wild type cells. This strongly suggests that mutant POT1 causes chromosome instability and may play a role in lymphomagenesis in these families.

Keywords: POT1; Hodgkin lymphoma; genetic analysis; genomic instability; telomere.

Figure 1

Hodgkin Lymphoma Families with POT1 mutations

Figure 2

a. Electrophoretic mobility shift assay measures the binding of in vitro translated (i.v.t.) wild-type (WT) and mutant HA-hPOT1 to [p32]-labelled single-stranded telomere oligonucleotides (GGTTAG)2. The amount of i.v.t. hPOT1 for the electrophoretic mobility shift assay was determined by Western blot using the anti-Flag antibody. b–e. Telomere length and Fragility of POT1 p.D224N and POT1 WT. For comparison purposes, binding to POT1 p.S270N which we reported previously (Shi, et al 2014), is shown. b, d. Telomere restriction fragment analysis of HT1080 cells using Biotin-labelled telomere probe. Telomere length is demonstrated by telomere signal intensities at higher and lower molecular weights. c, e. Telomere-FISH analysis of HT1080 cells expressing WT and mutant Flag-POT1 at indicated passages. c. Representative metaphase spread of a lymphoblastoid cell expressing either POT1 WT or p.D224N showing 4′,6-diamidino-2-phenylindole (DAPI) (grey) and telomere (red) fluorescence signals. Arrows indicate fragile telomeres (enlarged view in inset; 2.5× magnification). e. Quantitative measurement of telomere signal intensity in a plot displaying the distribution of telomeres with different signal intensities. Telomere signal intensity is depicted in arbitrary units (A.U.). Mean value denotes the mean telomere signal intensities in the indicated samples. Error bars are shown as mean ± standard deviation. Percentage of fragile telomeres was obtained from at least 30 metaphase spreads per sample. Error bar represents standard error of the mean. P values were obtained from the Wilcoxon rank-sum test. n.s.: not significant.