Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease

© 2014 John Wiley & Sons Ltd. Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P  <  0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI]  = 11·6-106·4) and this was preserved in early-stage disease patients (P  <  0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL

Telomere instability in the male germline

Telomeres play a key role in upholding the integrity of the genome, and telomerase expression in spermatogonial stem cells is responsible for the maintenance of telomere length in the human male germline. We have previously described extensive allelic variation in somatic cell telomere length that is set in the zygote, the ultimate source of which may be the germline. This implies that despite telomerase activity, substantial telomere length variation can be generated and tolerated in the germline; in order to investigate this further, we have examined the nature of telomere length variation in the human male germline. Here, we describe an analysis of both genome-wide telomere length and single molecule analysis of specific chromosome ends in human sperm. We observed individual specific differences in genome-wide telomere length. This variation may result from genetic differences within the components that determine the telomere length setting of each individual. Superimposed on the genome wide telomere length setting was a stochastic component of variation that generates germ-cells containing severely truncated telomeres. If not re-lengthened during early embryogenesis, such telomeres may limit the replicative capacity of cells derived from the zygote and have the potential to create fusagenic chromosomes, unbalanced translocations and terminal micro-deletions. These data may have implications for the genetic determination of ageing, genetic disease and fertility

Impact of sequence variation in the ul128 locus on production of human cytomegalovirus in fibroblast and epithelial cells

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism

Mre11 modulates the fidelity of fusion between short telomeres in human cells

The loss of telomere function can result in the fusion of telomeres with other telomeric loci, or non-telomeric double-stranded DNA breaks. Sequence analysis of fusion events between short dysfunctional telomeres in human cells has revealed that fusion is characterized by a distinct molecular signature consisting of extensive deletions and micro-homology at the fusion points. This signature is consistent with alternative error-prone end-joining processes. We have examined the role that Mre11 may play in the fusion of short telomeres in human cells; to do this, we have analysed telomere fusion events in cells derived from ataxia-telangiectasia-like disorder (ATLD) patients that exhibit hypomorphic mutations in MRE11. The telomere dynamics of ATLD fibroblasts were indistinguishable from wild-type fibroblasts and they were proficient in the fusion of short telomeres. However, we observed a high frequency of insertion of DNA sequences at the fusion points that created localized sequence duplications. These data indicate that Mre11 plays a role in the fusion of short dysfunctional telomeres in human cells and are consistent with the hypothesis that as part of the MRN complex it serves to stabilize the joining complex, thereby controlling the fidelity of the fusion reaction

Extensive telomere erosion is consistent with localised clonal expansions in Barrett’s metaplasia

Barrett’s oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett’s oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett’s oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett’s metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett’s metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett’s metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett’s metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett’s metaplasia

HCMV Spread and Cell Tropism are Determined by Distinct Virus Populations

Human cytomegalovirus (HCMV) can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A) shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A) complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC) cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A) in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells

Fusion of short telomeres in human cells is characterized by extensive deletion and microhomology, and can result in complex rearrangements

Telomere fusion is an important mutational event that has the potential to lead to large-scale genomic rearrangements of the types frequently observed in cancer. We have developed single-molecule approaches to detect, isolate and characterize the DNA sequence of telomere fusion events in human cells. Using these assays, we have detected complex fusion events that include fusion with interstitial loci adjacent to fragile sites, intra-molecular rearrangements, and fusion events involving the telomeres of both arms of the same chromosome consistent with ring chromosome formation. All fusion events were characterized by the deletion of at least one of the telomeres extending into the sub-telomeric DNA up to 5.6 kb; close to the limit of our assays. The deletion profile indicates that deletion may extend further into the chromosome. Short patches of DNA sequence homology with a G:C bias were observed at the fusion point in 60% of events. The distinct profile that accompanies telomere fusion may be a characteristic of the end-joining processes involved in the fusion event

BST2/Tetherin Enhances Entry of Human Cytomegalovirus

Interferon-induced BST2/Tetherin prevents budding of vpu-deficient HIV-1 by tethering mature viral particles to the plasma membrane. BST2 also inhibits release of other enveloped viruses including Ebola virus and Kaposi's sarcoma associated herpesvirus (KSHV), indicating that BST2 is a broadly acting antiviral host protein. Unexpectedly however, recovery of human cytomegalovirus (HCMV) from supernatants of BST2-expressing human fibroblasts was increased rather than decreased. Furthermore, BST2 seemed to enhance viral entry into cells since more virion proteins were released into BST2-expressing cells and subsequent viral gene expression was elevated. A significant increase in viral entry was also observed upon induction of endogenous BST2 during differentiation of the pro-monocytic cell line THP-1. Moreover, treatment of primary human monocytes with siRNA to BST2 reduced HCMV infection, suggesting that BST2 facilitates entry of HCMV into cells expressing high levels of BST2 either constitutively or in response to exogenous stimuli. Since BST2 is present in HCMV particles we propose that HCMV entry is enhanced via a reverse-tethering mechanism with BST2 in the viral envelope interacting with BST2 in the target cell membrane. Our data suggest that HCMV not only counteracts the well-established function of BST2 as inhibitor of viral egress but also employs this anti-viral protein to gain entry into BST2-expressing hematopoietic cells, a process that might play a role in hematogenous dissemination of HCMV

Human cytomegalovirus infection of langerhans-type dendritic cells does not require the presence of the gH/gL/UL128-131A complex and is blocked after nuclear deposition of viral genomes in immature cells

Human cytomegalovirus (CMV) enters its host via the oral and genital mucosae. Langerhans-type dendritic cells (LC) are the most abundant innate immune cells at these sites, where they constitute a first line of defense against a variety of pathogens. We previously showed that immature LC (iLC) are remarkably resistant to CMV infection, while mature LC (mLC) are more permissive, particularly when exposed to clinical-strain-like strains of CMV, which display a pentameric complex consisting of the viral glycoproteins gH, gL, UL128, UL130, and UL131A on their envelope. This complex was recently shown to be required for the infection of immature monocyte-derived dendritic cells. We thus sought to establish if the presence of this complex is also necessary for virion penetration of LC and if defects in entry might be the source of iLC resistance to CMV. Here we report that the efficiency of LC infection is reduced, but not completely abolished, in the absence of the pentameric complex. While virion penetration and nuclear deposition of viral genomes are not impaired in iLC, the transcription of the viral immediate early genes UL122 and UL123 and of the delayed early gene UL50 is substantially lower than that in mLC. Together, these data show that the UL128, UL130, and UL131A proteins are dispensable for CMV entry into LC and that progression of the viral cycle in iLC is restricted at the step of viral gene expression