Abstract Missense mutations in TERT , the gene encoding the human telomerase catalytic subunit hTERT, are associated with Telomere Biology Disorders (TBDs). Experimentally elucidating the effects of all possible missense variants would be time-consuming and technically challenging. Moreover, current computational predictors are not hTERT-specific and primarily rely on sequence information, failing to capture the complex biological and structural context of the telomerase enzyme. In this work, we developed three machine learning models integrating both sequence- and structure-based features to account for the biological mechanisms of hTERT. Compared to state-of-the-art methods, our best-performing models achieved a higher Matthew’s Correlation Coefficient of 0.88 on ClinVar and gnomAD curated variants and demonstrated robust sensitivity (0.75) on a dataset curated according to guidelines from the American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG/AMP). Feature interpretation highlighted hTERT residue conservation and changes in hydrophobic and weak polar interactions as critical determinants of pathogenicity. Finally, in silico saturation mutagenesis was performed to present a mutational landscape of TERT , available in a user-friendly web server, CharacTERT, which could offer valuable insights into the molecular mechanisms driving TBDs, aid in early diagnosis, as well as guide personalized treatment strategies. CharacTERT is freely available at https://biosig.lab.uq.edu.au/charactert/ .

Rare single base pair changes in genes are an important cause of disease, as they can reside in key regions of the gene influencing biological function by impacting the protein conformation and protein interactions. Generation of the necessary experimental evidence to define the outcome of the presence of these gene variants is time-consuming and costly. These challenges have led to the development of a plethora of in silico prediction tools. These tools frequently use similar sources of information and are trained on overlapping multigene 'truth' datasets. However, frequently there has been no quantitative validation of the performance of these in silico tools for individual genes. Here, we have applied the ClinGen Sequence Variant Interpretation Working Group's recommended in silico score thresholds and AlphaMissense predictions to a set of predisposition gene variants with established pathogenicity/benignity. Of the genes assessed (BRCA1, BRCA2, TP53, TERT and ATM), when recommended thresholds were used, in silico tool predictions showed inferior sensitivity (< 65%) for pathogenic TERT variants and inferior sensitivity (≤ 81%) for benign TP53 variants. AlphaMissense outperformed the other tools for TP53 but did not improve predictive accuracy for TERT variants. This validation study highlights that in silico tool performance can be gene-specific and is dependent on the 'training set' on which the algorithm is built. Where there are sufficient numbers of established benign and pathogenic missense variants based on clinical and functional evidence, the use of in silico tool scores should be validated for individual genes. For genes where this is not possible and gene-agnostic in silico score cut-offs are used, consideration of missense variant-protein structural impact relationships is suggested.

Telomerase is a ribonucleoprotein enzyme that maintains telomeric repeats on chromosome ends in continuously dividing cells. Telomere maintenance via telomerase is dependent on the correct assembly of the enzyme complex, complex stabilization by associated cofactors, and effective recruitment to the telomere. Here, we show that telomerase is regulated in each of these processes by the Drosophila behaviour/human splicing (DBHS) family of RNA/DNA binding proteins (NONO, SFPQ and PSPC1). The DBHS proteins associate with catalytically active telomerase through the hTR RNA template component. Cells lacking the DBHS proteins display telomerase retention in nuclear Cajal bodies and impaired telomerase recruitment to the telomere, with NONO and PSPC1 depletion culminating in progressive telomere shortening in several cell lines, with the exception of long-term NONO depletion in 293 and 293T. Our results reveal the DBHS protein family as components of the telomerase trafficking machinery integral to telomere maintenance.

The recruitment of telomerase to telomeres is a tightly regulated process which is stimulated by replication stress and the DNA damage response regulatory kinase ATR, via an unknown mechanism. Here, we demonstrate that nuclear filamentous actin is important for the stable interaction of telomerase with telomeres in immortal human cells, resulting in productive telomere elongation by telomerase in an actin-dependent manner. This process is regulated by both ATR and mTOR kinases, and employs other regulators of actin structure and function, such as WASP, ARP2/3 and myosin. Nuclear filamentous actin serves as a site for telomerase recruitment, which is mediated by telomere tethering on actin fibers in response to replication stress, allowing telomerase to localize to telomeres containing stalled replication forks. Overall, these data demonstrate that, in human cells which express telomerase, telomeric replication stress triggers the recruitment of telomerase to telomeres via a nuclear actin network, enabling telomere length maintenance.

Abstract Inherited bone marrow failure syndromes (IBMFS) are a group of monogenic diseases of diverse pathogenesis manifesting as single or multilineage cytopenia typically due to hypoproliferative dyshematopoiesis. Accurately diagnosing IBMFS is challenging given the overlapping clinicopathological features between individual genetic syndromes as well as with acquired BMFS (e.g. immune aplastic anemia). Accurate genetic diagnosis in IBMFS is critical and most commonly involves targeted panel DNA sequencing or whole exome sequencing. These approaches do not cover the full spectrum of possible genomic abnormalities which in turn may contribute to a significant proportion of patients with IBMFS remaining undiagnosed. We aimed to comprehensively evaluate the unbiased upfront approach of whole genome transcriptome sequencing (WGTS) in patients with suspected IBMFS (the IBMDx study). The IBMDx study aimed to (i) determine the diagnostic rate and clinical impact of upfront WGTS (ii) assess the acceptability of WGTS to patients and physicians through an implementation science framework (iii) evaluate the health-economic impact and cost-effectiveness of the approach and (iv) discover and functionally characterize novel genes and variants in IBMFS. 237 patients were enrolled from March 2022 to March 2025. The median age of the cohort was 30 years (range 6m-78 years; M:F 0.93:1). 70/237 (29.5%) were under 18 years. WGS was performed exclusively on non-hematological DNA (hair follicle DNA [n=178], skin biopsy/cultured skin fibroblasts [n=59]). Trio WGS was performed in 18 families. WGS was performed by a clinically accredited service and results returned to physicians/patients in real time for patient management and segregation/predictive testing as required. A genomic diagnosis for the hematological phenotype was established in 88/237 (37.1%) patients. Diagnoses made included hereditary thrombocytopenia (n=19), telomere biology disorders (TBDs) (n=14), Diamond-Blackfan anemia (n=12), primary red cell disorders (n=6), severe congenital neutropenia (n=6), Shwachman Diamond Syndrome (n=5) and Fanconi anemia (n=4). Targeted sequencing of the hematological compartment demonstrated clonal hematopoiesis in 32/217 (14.7%) patients. In addition, four patients had genetic diagnoses made of symptomatic and clinically significant diseases unrelated to the hematological phenotype (TAP2, IRF2BP2, ACADM and mosaic trisomy 7). Secondary genomic findings requiring further management were detected in 7 patients (TNNI3, MSH6, Monosomy X mosaic, BRCA1, BRIP1, ATM, FBN1). WGTS revealed novel genomic abnormalities not previously established in IBMFS such as retrotransposon-mediated gene disruption, polyadenylation site loss and disruption of novel regulatory regions. Novel genomic abnormalities in genes associated with Diamond-Blackfan anaemia (RPL31), TBDs (TERT/TERC) and hereditary thrombocytopenia (TPM4) underwent in vivo and in vitro functional assessment to inform pathogenicity. Finally, novel associations were identified with primary IBMFS-like presentations (THRA) as well as definitive evidence of gene-disease association were established (MEIS1, TUBB). Using a formal Theoretical Framework of Acceptability, WGTS was found to be highly acceptable to patients and carers as evidenced by positive perceptions of clinicians, alignment with healthcare expectations, and minimal effort required to participate. However, concern regarding equity of access to technology for all patients with suspected IBMFS was a recurrent theme amongst patients and carers interviewed. Health economic analysis showed that the cost of achieving maximum diagnostic yield, incorporating varied diagnostic strategies, to be between $7,800 - $8,200 USD per hematological diagnosis. In summary, we have comprehensively evaluated upfront WGTS in a large cohort of adult and pediatric patients with suspected IBMFS and have found this approach has a high diagnostic rate (37.1%), is highly acceptable to patients, and uncovered multiple genomic abnormalities that would have been missed with more traditional diagnostic approaches. Moreover, we have uncovered new genomic mechanisms of disease and genes associated with IBMFS leading to new areas of research into the underlying biology of these challenging diseases.

Pathogenic germline variants in telomerase (TERT) cause telomere biology disorders (TBDs) and are associated with bone marrow failure, pulmonary fibrosis, and other complications. TERT c.3150 G > C (p.K1050N) is frequent in the Ashkenazi Jewish (ASH) population and has been identified in ASH families with TBDs. Whole-genome sequencing of 96 p.K1050N heterozygotes from the UK Biobank and All of Us databases revealed a shared haplotype block, supporting a founder effect. Analyses of 15 additional p.K1050N cases validated this haplotype and identified mitochondrial and Y-STR haplogroups consistent with ASH ancestry. Clinical assessments showed that p.K1050N contributes to TBD phenotypes and shortened telomeres, while population data suggest incomplete penetrance. p.K1050N reduces telomerase activity and processivity, and decreases PCNA expression and BrdU incorporation, impairing cell proliferation. Our findings establish TERT p.K1050N as an ASH founder variant associated with TBDs, underscoring the need for genetic screening and long-term clinical studies.

Telomere biology disorders (TBDs) are inherited conditions associated with multisystem manifestations. We describe clinical and functional characterisation of a novel TERT variant. Whole-genome sequencing was performed along with single telomere length analysis (STELA). Telomerase activity and processivity were assessed. A novel TERT variant (K710R) was detected in a patient with classic TBD features showing reduced telomerase activity and processivity. Despite clinical and functional evidence, the variant was classified as a variant of uncertain significance. We have described a novel TERT variant and highlighted the need for further refinement of variant classification specific for TBDs.

Cancer remains a leading cause of death worldwide and although prognosis and survivorship after therapy have improved significantly, current cancer treatments have long-term health consequences. For decades telomerase-mediated telomere maintenance has been an attractive anti-cancer therapeutic target due to its abundance and role in telomere maintenance, pathogenesis, and growth in neoplasms. Telomere maintenance-specific cancer therapies, however, are marred by off-target side effects that must be addressed before they reach clinical practice. Regular exercise training is associated with telomerase-mediated telomere maintenance in normal cells, which is associated with healthy aging. A single bout of endurance exercise training dynamically, but temporarily, increases TERT mRNA and telomerase activity, as well as several molecules that control genomic stability and telomere length (i.e., shelterin and TERRA). Considering the epidemiological findings and accumulating research highlighting that exercise significantly reduces the risk of many types of cancers and the anti-carcinogenic effects of exercise on tumor growth in vitro, investigating the governing molecular mechanisms of telomerase control in context with exercise and cancer may provide important new insights to explain these findings. Specifically, the molecular mechanisms controlling telomerase in both healthy cells and tumors after exercise could reveal novel therapeutic targets for tumor-specific telomere maintenance and offer important evidence that may refine current physical activity and exercise guidelines for all stages of cancer care.

The recruitment of telomerase to telomeres is a tightly regulated process which is stimulated by replication stress and mediated by the DNA damage response regulatory kinase ATR. Here, we demonstrate that nuclear filamentous actin is important for telomerase recruitment under endogenous and replication stress conditions in immortal human cells. Inhibition of nuclear actin polymerization decreases the presence of telomerase at telomeres. This process is regulated by both ATR and mTOR kinases, and employs other regulators of actin structure and function, such as WASP, ARP2/3 and myosin. Nuclear filamentous actin serves as a site for telomerase recruitment, which is mediated by telomere tethering on actin fibres in response to replication stress, allowing telomerase to localize to telomeres containing stalled replication forks. Overall, these data demonstrate that, in human cells which express telomerase, telomeric replication stress triggers the recruitment of telomerase to telomeres via a nuclear actin network, enabling telomere length maintenance.### Competing Interest StatementThe authors have declared no competing interest.

Telomeres are the protective caps at the ends of linear chromosomes of eukaryotic organisms. Telomere binding proteins, including the six components of the complex known as shelterin, mediate the protective function of telomeres. They do this by suppressing many arms of the canonical DNA damage response, thereby preventing inappropriate fusion, resection and recombination of telomeres. One way this is achieved is by facilitation of DNA replication through telomeres, thus protecting against a “replication stress” response and activation of the master kinase ATR. On the other hand, DNA damage responses, including replication stress and ATR, serve a positive role at telomeres, acting as a trigger for recruitment of the telomere-elongating enzyme telomerase to counteract telomere loss. We postulate that repression of telomeric replication stress is a shared mechanism of control of telomerase recruitment and telomere length, common to several core telomere binding proteins including TRF1, POT1 and CTC1. The mechanisms by which replication stress and ATR cause recruitment of telomerase are not fully elucidated, but involve formation of nuclear actin filaments that serve as anchors for stressed telomeres. Perturbed control of telomeric replication stress by mutations in core telomere binding proteins can therefore cause the deregulation of telomere length control characteristic of diseases such as cancer and telomere biology disorders.

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

Supplementary Figure S7. OTX015 and carfilzomib synergistically improve mouse survival in a PDX model of TERT-rearranged neuroblastoma.

Supplementary Figure S5. BET bromodomain inhibitors and proteasome inhibitors exert synergistic anticancer effects against TERT-rearranged neuroblastoma cells.

Supplementary Figure S2. BRD4 is required for TERT expression and cell proliferation in TERT-rearranged neuroblastoma cells.

Supplementary Figure S4. Combination of the BET bromodomain inhibitor I-BET762 and chemotherapy agents, but not proteasome inhibitors, induces cytotoxicity to normal cells.

Supplementary Methods and Materials from Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

Telomere length is an important determinant of cellular aging and disease risk, but the genetics of telomere length control in humans is unclear. A genome-wide CRISPR screen has now identified a central role for thymidine nucleotide metabolism in the regulation of telomere length, which has implications for the diagnosis and treatment of disease.

Re - Revised Supplementary Materials & Methods

Telomeres are specialized, highly conserved DNA-protein complexes at the ends of linear eukaryotic chromosomes. Human telomeric DNA is composed of tandem repeats of the sequence 5′-(TTAGGG)n-3′ and is complexed with sequence-specific DNA binding proteins, forming a distinctive "cap" at the ends of chromosomes. Telomeres serve to distinguish the end of the chromosome from an internal DNA break and the cellular DNA repair machinery, thereby protecting chromosomes from deleterious end-to-end fusions. Telomeres are dynamic structures, shortening during each cycle of DNA replication and cell division. In the absence of a compensatory telomere-lengthening mechanism, progressive telomere shortening imposes limits on the proliferative capacity of cells, contributing to organismal aging. Cells with unlimited proliferative capacity – notably stem cells and cancer cells – must activate a telomere lengthening mechanism. There are two such mechanisms known in humans: the ribonucleoprotein enzyme telomerase (discussed in Chap. Telomerase) and the recombination-based mechanism Alternative Lengthening of Telomeres. Most human somatic cells do not maintain their telomeres and undergo telomere shortening. In contrast, dysregulated telomere maintenance is a universal property of cancer cells. Understanding the biology and dynamics of telomeres has far-reaching implications for human health and medicine.

Supplementary Figure S6. OTX015 and carfilzomib exert synergistic anticancer effects partly by inducing oxidative stress and endoplasmic reticulum stress.