Telomere Biology Genes Research Articles

Telomere transcripts act as tumor suppressor and are associated with favorable prognosis in colorectal cancer with low proliferating cell nuclear antigen expression.

Telomeric repeat-containing RNAs (TERRA) and telomerase RNA component (TERC) regulate telomerase activity (TA) and thereby contribute to telomere homeostasis by influencing telomere length (TL) and the cell immortality hallmark of cancer cells. Additionally, the non-canonical functions of telomerase reverse transcriptase (TERT) and TERRA appear to be involved in the epithelial-mesenchymal transition (EMT), which is important for cancer progression. However, the relationship between TERRA and patient prognosis has not been fully characterized. In this small-scale study, 68 patients with colorectal cancer (CRC) were evaluated for correlations between telomere biology, proliferation, and EMT gene transcripts and disease outcome. The proliferating cell nuclear antigen (PCNA) and the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) showed a positive correlation with TERRA, while TA and TERRA exhibited an inverse correlation. Consistent with previous findings, the present study revealed higher expression levels of TERT and TERC, and increased TA and TL in CRC tumor tissue compared to adjacent non-tumor tissue. In contrast, lower expression levels of TERRA were observed in tumor tissue. Patients with high TERRA expression and low PCNA levels exhibited favorable overall survival rates compared to individuals with the inverse pattern. Furthermore, TERRA suppressed CRC tumor growth in severe combined immunodeficiency disease (SCID) mice. In conclusion, our study extends previously published research on TERRA suggesting its potential therapeutic role in telomerase-positive CRC.

Genetics and Genomics of Pulmonary Fibrosis: Charting the Molecular Landscape and Shaping Precision Medicine.

Recent genetic and genomic advancements have elucidated the complex etiology of idiopathic pulmonary fibrosis (IPF) and other progressive fibrotic interstitial lung diseases (ILDs), emphasizing the contribution of heritable factors. This state-of-the-art review synthesizes evidence on significant genetic contributors to pulmonary fibrosis (PF), including rare genetic variants and common SNPs. The MUC5B promoter variant is unusual, a common SNP that markedly elevates the risk of early and established PF. We address the utility of genetic variation in enhancing understanding of disease pathogenesis and clinical phenotypes, improving disease definitions, and informing prognosis and treatment response. Critical research gaps are highlighted, particularly the underrepresentation of non-European ancestries in PF genetic studies and the exploration of PF phenotypes beyond usual interstitial pneumonia/IPF. We discuss the role of telomere length, often critically short in PF, and its link to progression and mortality, underscoring the genetic complexity involving telomere biology genes (TERT, TERC) and others like SFTPC and MUC5B. In addition, we address the potential of gene-by-environment interactions to modulate disease manifestation, advocating for precision medicine in PF. Insights from gene expression profiling studies and multiomic analyses highlight the promise for understanding disease pathogenesis and offer new approaches to clinical care, therapeutic drug development, and biomarker discovery. Finally, we discuss the ethical, legal, and social implications of genomic research and therapies in PF, stressing the need for sound practices and informed clinical genetic discussions. Looking forward, we advocate for comprehensive genetic testing panels and polygenic risk scores to improve the management of PF and related ILDs across diverse populations.

Bone Marrow Failure Associated with Short Telomeres and Digenic Variants of Uncertain Significance in Telomere Biology Genes

DISCUSSION Telomere Biology Disorders (TBD) are a group of heritable disorders characterized by abnormally short telomeres. There are at least 15 genes associated with TBD including ACD, CTC1, DKC1, NHP2, NOP10, PARN, POT1, RTEL1, STN1, TERC, TERT, TINF2, WRAP53 NAF1 and ZCCHC8, which can be inherited in autosomal dominant (AD), autosomal recessive (AR), or X-linked recessive patterns. Genetic anticipation has been described in some families with more severe symptoms appearing in each successive generation. We report two patients that presented with bone marrow failure, who were subsequently identified to have short telomeres and variants of uncertain significance (VUS) in two different telomere genes inherited from their parents. These cases suggest a potential for digenic inheritance of TBD. CASE 1: At age 6, patient was diagnosed with stage 4 neuroblastoma. He was treated with chemotherapy, surgery, immunotherapy, and autologous stem cell rescue. At age 10, he developed frequent and severe epistaxis and was found to have pancytopenia. He underwent a bone marrow aspirate and biopsy which revealed a hypocellular marrow and der (1;7), which is associated with myelodysplastic syndrome (MDS). Genetic evaluation revealed one pathogenic variant in DNAJC21 (from father) and VUS in NAF1 (c.1375C>T) (from father) and RTEL1 (c.533T>C) (from mother). Because NAF1 and RTEL1 are related to AD or AR TBD, telomere length testing was performed. Patient was found to have low telomere length, < 1st percentile, in granulocytes, lymphocytes, memory T cells and NK cells (Figure 1). The patient's mother was found to have borderline low telomere length >=1 and <10 th percentiles in her lymphocytes, naïve T cells, memory T cells, and B cells, and low telomere length in her granulocytes. Father had borderline low telomere length >=1 and <10 th percentiles in granulocytes but normal telomere length in all other subsets. Neither parent reported any symptoms that would be consistent with TBD. We interpreted these findings as consistent with the patient having TBD and the patient's mother either also having TBD or being a carrier, and the potential that genetic anticipation may be involved. The NAF1 variant appears likely to be benign as father's telomeres were normal in length except for low telomere length in his granulocyte compartment. It is possible that the patient's prior treatment for neuroblastoma may have contributed to the shortened telomeres. Over the course of a year, patient developed worsening bone marrow failure. He then underwent reduced intensity conditioning using busulfan, fludarabine and anti-thymocyte globulin for a 4/6 matched cord stem cell transplant. He engrafted on day +10. CASE 2: A 15-year-old male presented with a depressed skull fracture and scalp hematoma following a sports-related head injury. He was incidentally found to have a platelet count of 69 x 10 3/microliter, mild anemia with macrocytosis, and mild leukopenia. Bone marrow biopsy revealed a hypocellular marrow with multilineage maturation, mild fibrosis, and no cytogenetic abnormalities. The patient's telomere lengths were low, below the 1 st percentile, in all 6 white blood cell subsets (Figure 2). He underwent clinical trio exome sequencing along with his parents. He was found to have VUSs in TERT (c.3158-80G>A) (from mother), TINF2 (c.814T>C) (from mother) and SRP72 (c.1928C>T) (from father). Mother was also found to have low telomere length (<1 st percentile) in all 6 subsets. Both parents have normal blood counts while patient continues to have pancytopenia and is transfusion independent. It is presently unclear which of the VUSs are contributing to the patient's phenotype as the TERT and TINF2 variants are both associated with bone marrow failure and heterozygous germline variants in SRP72 is associated with familial MDS. Genes that cause TBD can be inherited through various modes of inheritance. These two patients who presented with bone marrow failure, identified to have VUSs in more than one TBD-associated genes and functional evidence of shortened telomeres, highlight the potential for a digenic mode of inheritance underlying TBD. Synergy between two variants contributes to a penetrant phenotype and can result in earlier or more severe onset of disease. Our patients' parents were asymptomatic or mildly affected, compared to our patients. Larger cohort studies are needed to determine the prevalence of this mode of inheritance.

Immune Signature in Telomere-Biology Disorders

Excessive telomere shortening caused by pathogenic germline variants in telomere-biology genes may result in bone marrow failure, hematopoietic malignancy and extramedullary complications, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, and solid tumors. Patients with short telomeres also may develop immunodeficiency with low counts of T-cell receptor excision circles (TRECs), kappa-deleting excision circles (KRECs), and CD4 + T cells. The aim of the present study was to comprehensively determine the immune system changes in human patients with telomeropathies by deep-phenotyping peripheral blood mononuclear cells (PBMCs) and quantifying cytokine serum levels. We applied a 39-marker panel of metal-labeled antibodies and employed mass cytometry (CyTOF) to assess the subsets of lymphocytes, monocytes, and dendritic cells (DC) in 20 patients and 10 healthy individuals. High-dimensional analysis was conducted using automated clustering ( Flow-SOM) to identify subpopulations and cell states, whereas t-SNE was employed as a visualization tool. A total of 32 serum cytokines, chemokines and growth factors were quantified by a bead-based immunoassay (Luminex™). First, we confirmed previous findings of low CD4/CD8 ratio (0.73±0.56 vs. 1.3±0.69 in controls, p=0.05), and reduced absolute naïve (CD45RA +CCR7 +) CD4 + and CD8 + cell counts ( p<0.01), with effector memory cell accumulation in patients with telomere diseases, and the current study further showed a reduction in naïve B (CD19 +IgD +CD27 -) and in recent thymic emigrants (RTE), characterized by CD31 +CD45RA + ( p<0.01), indicating a consistent aged B and T cell profile. T H1, T H17 and T H17.1 CD4 + lymphocytes were reduced ( p<0.05), whereas the T H2/T H1 ratio was increased (1.6±0.83 vs. 0.82±0.39 in controls; p=0.013), demonstrating a dysregulated T helper cell diversity. T regs, on the other hand, did not appear to be affected. Plasmacytoid DCs (CD3 -CD141 +CD123 +CD1c -), which are known inducers of T helper differentiation, were reduced in patients ( p=0.014). A double negative T cell (DNT) population, characterized as CD3 +CD4 -CD8 -Vd2 -Tbet +Granzyme B +, was significantly increased ( p=0.0002). Activated effector memory and TEMRA CD4 + cells, also expressing exhaustion markers PD-1 and TIGIT (CD4 +CD45RA +/-CCR7 -CD38 +HLADR +Ki67 +PD-1 +TIGIT low) and proliferating switched memory B cells (CD3 -CD19 +IgD -CD27 +Ki67 +) were higher ( p=0.046 and p=0.041, respectively) suggesting the occurrence of an environment prone to immune activation, also supported by the prevalence of CD14 +CD16 +CXCR3 +CCR4 + classical monocytes ( p=0.0042). Additionally, mucosal-associated invariant T (MAIT) cells (CD3 +CD161 highVɑ7.2 +) were markedly reduced in patients ( p<0.0001), possibly due to their recruitment to other tissues as a response to inflammation. Conversely, immature NK cells (CD3 -CD19 -CD56 brightCD16 -) were more frequent in patients as compared to controls ( p=0.026), and individuals with the shortest telomere lengths had an even higher frequency of these cells. In addition to the previously described CD4 + immunodeficiency, the predominance of immature NK cells may potentially impact the immunosurveillance of malignant transformations. When an unsupervised clustering algorithm using cell subpopulations as variables was applied, patients and controls were correctly identified in 100% of cases. Telomere length positively correlated with some cytokine levels in serum. This was observed for angiopoietin-1, IL-1 superfamily, IL-3, IL-4, IL-7, IL-23, IL-27, M-CSF, MCP-1, MIP-3a and PDGF-BB. Taken together, our results reveal a distinct immunophenotype in telomeropathies, which comprises deficient or skewed maturation of certain subsets, an aged phenotype of B and T cells, aberrant lymphocyte activation and very low levels of circulating MAIT cells, which might be indicators of ongoing inflammatory processes, tied with dysregulated cytokine levels. This immune perturbance may contribute to the clinical onset of telomeropathies and increase the risk of infections, cirrhosis, IPF and cancer development.

Contribution of Genetic Screening to the Diagnosis of Patients with Bone Marrow Failure and Short Telomeres

Telomeres are structures responsible for the maintenance of chromosomal stability. Telomere length is maintained by the telomerase complex, shelterin proteins, and others involved in telomere biology. Germline variants in telomere-biology genes may cause excessive telomere shortening, cell senescence, and genomic instability, which in humans may clinically translate into bone marrow failure, liver and lung fibrosis, skin lesions, and a proclivity for cancer. The prevalence of telomere diseases is unknown, partly due to variable penetrance. Patients without phenotypical signs or a family history may be misdiagnosed as having acquired aplastic anemia (AAA). Other inherited bone marrow failure syndromes (IBMF) and AAA may also present with short telomeres. The aim of this study was to determine the genetic variants in patients with bone marrow failure (BMF) and short telomeres. The study was conducted at a tertiary hospital in Sao Paulo, Brazil. One-hundred forty-four patients with BMF followed between 2016 and 2022 were enrolled. Ethical approval was obtained from the local Ethics Committee. Telomere length was determined by Southern blot or flow-FISH in peripheral blood leukocytes. Telomeres were interpreted as short when below the 10th percentile according to age. All samples were submitted to genomic analysis by Next Generation Sequencing (NGS), using a panel for 154 genes associated with hematologic malignancies and BMF syndromes. A presumptive diagnosis was made based on clinical and laboratorial data. Liver or pulmonary fibrosis, early greying, dystrophic nails, leukoplakia, hypopigmented skin, solid malignant tumors, retinopathy, family history of hematological diseases and consanguinity were considered as red flag symptoms for telomeropathies. Patients with no red flags who had severe cytopenias were considered as having possible AAA. Patients with non-severe cytopenias starting at an early age were considered as possibly having other IBMF syndromes. Thirty patients (21%) out of 144 analyzed had short telomere length and were included. They had their telomeres measured at diagnosis (53%) or at follow-up (47%).Combining clinical, laboratory, and NGS data, 10 patients (34%) were diagnosed with Telomeropathies [TERT (N=4), RTEL1 (N=3), DKC1 (N=1), TERC (N=1), ACD (N=1)]. Six patients (20%) were diagnosed with other IBMF syndromes [4 Fanconi anemia (FANCA, FANCG, BRCA2), 1 GATA2 deficiency (GATA2), 1 Blackfan-Diamond anemia (RPS19)]. Seven patients (23%) were considered as having AAA, not having any germline variants, and 7 (23%) as undetermined cases, with some suggestive clinical feature but no identified germline variant (figure 1). Comparing with NSG data, the clinical and laboratorial presumptive diagnosis had a 95% accuracy, 100% sensitivity and 92% specificity. In the group with confirmed telomeropathy (N=10), 70% were male, 80% were white, and the median age at diagnosis was 25 years. All of them had at least one red flag symptom: hepatic fibrosis (60%), early graying (60%), family history of hematologic diseases (40%) and one patient had the classic dermatologic triad of dyskeratosis congenital. Only one had severe cytopenia and none had significant paroxysmal nocturnal hemoglobinuria clone. Most of them had hypoplastic bone marrow without significant dysplasia (N=8). In conclusion, clinical features combined with laboratory analysis had high accuracy when compared with NGS. Despite having access to NGS, a final diagnosis was not achieved for 23% of patients, who would benefit from further genetic analysis.

Abstract 4108: Understanding the role of telomere biology gene variation in cancer etiology

Abstract Telomere biology disorders (TBDs) are cancer-prone syndromes associated with increased risk of acute myeloid leukemia (AML), head and neck squamous cell carcinoma (HNSCC), bone marrow failure, pulmonary fibrosis, and liver disease. Germline variants in at least 15 different telomere biology genes have been implicated (ACD, CTC1, TERT, TERC, STN1, NAF1, NOP10, NHP2, TINF2, RTEL1, PARN, ZCCHC8, DKC1, WRAP53, POT1) with autosomal dominant (AD), autosomal recessive (AR), or X-linked (XLR) inheritance as well as de novo occurrence. We hypothesize that TBDs may be more common than the currently estimated 1:1,000,000. We analyzed the prevalence of germline variants in the 15 TBD-associated genes using 1) The Genome Aggregation Database [gnomAD v2.1.1 non-cancer] and 2) The Cancer Genome Atlas (TCGA). Germline TCGA variants were called using HaplotypeCaller, Freebayes, and UnifiedGenotyper. All variants with minor allele frequency (MAF) <1% in gnomAD non-cancer were considered. They were classified as potentially deleterious based on ClinVar and/or loss of function classification and/or stringent in silico predictions utilizing REVEL, MetaSVM, CADD, BayesDel, Eigen for missense and a combination of spliceAI, spidex, and dbscSNV for splice site variants. For TERC (RNA telomerase template) variants in the pseudoknot/template region were considered deleterious. The known disease-causing inheritance pattern (AD, AR, XLR) for each gene was used to identify TCGA cases with a probable TBD. There were 1215 potentially deleterious variants in TBD associated genes in the gnomAD v2.1.1 non-cancer dataset (n=134,187 samples) for a combined prevalence of 0.9%, without accounting for zygosity. In genes with AD/AR inheritance, variants were most common in: RTEL1 (249), TERT (104), and PARN (104). CTC1 and WRAP53, both associated with solely AR TBDs, also showed high variant frequencies (280 and 102, respectively). In the TCGA dataset, 9089 cancer cases (32 solid tumors and AML) were evaluated. We identified 227 cases (2.5%) with 161 mono- or biallelic rare, potentially deleterious variants in the 15 genes. Ninety-two cases had a probable underlying TBD [1% of total, 41 male, 51 female, median age at cancer diagnosis 46.5 (1-92) years]. Cancer diagnoses in these 92 cases included 24 solid tumors. The frequency of individuals with deleterious TBD-associated variants ranged from 0.4-2.8% in each of the 24 affected cancers. Notably, the heterozygous frequencies in lung squamous cell carcinoma and liver hepatocellular carcinoma were 1.6% and 2.2%, respectively. Our results show that the prevalence of TBDs may be noticeably higher than previously estimated, specifically in individuals with cancer. TBD-associated pathogenic germline variants may be implicated in the etiology of more solid tumor entities than previously recognized, warranting further studies. Citation Format: Marena R. Niewisch, Jung Kim, Judith C. Lunger, Lisa J. McReynolds, Sharon A. Savage. Understanding the role of telomere biology gene variation in cancer etiology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4108.

Disease progression and clinical outcomes in telomere biology disorders

Dyskeratosis congenita related telomere biology disorders (DC/TBDs) are characterized by very short telomeres caused by germline pathogenic variants in telomere biology genes. Clinical presentations can affect all organs, and inheritance patterns include autosomal dominant (AD), autosomal recessive (AR), X-linked (XLR), or de novo. This study examined the associations between mode of inheritance with phenotypes and long-term clinical outcomes. Two hundred thirty-one individuals with DC/TBDs (144 male, 86.6% known genotype, median age at diagnosis 19.4 years [range 0 to 71.6]), enrolled in the National Cancer Institute's Inherited Bone Marrow Failure Syndrome Study, underwent detailed clinical assessments and longitudinal follow-up (median follow-up 5.2 years [range 0 to 36.7]). Patients were grouped by inheritance pattern, considering AD-nonTINF2, AR/XLR, and TINF2 variants separately. Severe bone marrow failure (BMF), severe liver disease, and gastrointestinal telangiectasias were more prevalent in AR/XLR or TINF2 disease, whereas pulmonary fibrosis developed predominantly in adults with AD disease. After adjusting for age at DC/TBD diagnosis, we observed the highest cancer risk in AR/XLR individuals. At last follow-up, 42% of patients were deceased with a median overall survival (OS) of 52.8 years (95% confidence interval [CI] 45.5-57.6), and the hematopoietic cell or solid organ transplant-free median survival was 45.3 years (95% CI 37.4-52.1). Significantly better OS was present in AD vs AR/XLR/TINF2 disease (P < .01), while patients with AR/XLR and TINF2 disease had similar survival probabilities. This long-term study of the clinical manifestations of DC/TBDs creates a foundation for incorporating the mode of inheritance into evidence-based clinical care guidelines and risk stratification in patients with DC/TBDs. This trial was registered at www.clinicaltrials.gov as #NCT00027274.

Whole Exome Sequencing in Severe Aplastic Anemia Identifies Unrecognized Inherited Subset with Inferior Survival after Hematopoietic Cell Transplant

Background: It is important to identify inherited bone marrow failure syndromes (IBMFS) in patients with aplastic anemia in order to provide appropriate therapy. IBMFS are diagnosed through genetic or other diagnostic testing but can also go unrecognized, particularly in adults who may lack classic IBMFS features. This is particularly critical in light of increasing identification of individuals with variants of uncertain significance (VUS) and those with single pathogenic variants in an autosomal recessive (AR) gene or X-linked recessive (XLR) gene in females (SPVR) (“carriers”) during evaluation. In this study of patients diagnosed with acquired severe aplastic anemia (SAA), we evaluated putatively causal variants in IBMFS genes to determine the frequency of patients with an unrecognized IBMFS or SPVR and assessed their association with outcomes after hematopoietic cell transplant (HCT).Methods: We used pre-HCT blood samples and clinical data from the Transplant Outcomes in Aplastic Anemia study (TOAA; a collaboration between the National Cancer Institute and the Center for International Blood and Marrow Transplant Research). Germline whole exome sequencing was performed on 732 patients with acquired SAA who received HCT between 1991-2015. A total of 104 IBMFS genes, (51 autosomal dominant (AD), 46 AR, 3 both AR and AD, 4 XLR) were evaluated for both single nucleotide (SNV) and copy number variants (CNV). All variants were curated using ACMG/AMP criteria, and a subset were validated by Sanger sequencing. Variants classified as VUS according to ACMG/AMP criteria and with a damaging score prediction in 3 of 5 in silico meta-predictors were categorized as deleterious VUS. Patients were divided into 3 groups based on known inheritance patterns of identified genes into those with 1) unrecognized IBMFS 2) SPVR or 3) neither (presumed acquired SAA). For telomere biology genes with AD/AR inheritance, we used telomeres <10 th percentile for age (by qPCR) to distinguish IBMFS from SPVR. For statistical analysis, we used the Kaplan-Meier estimator to calculate the probability of overall survival. The log-rank test was used to compare the survival distribution across patient categories. Cox proportional hazard models were used for multivariable analysis.Results: We identified 309 variants of them 156 were pathogenic or likely pathogenic (P/LP) (112 (71.8%) loss of function SNVs, and 10 CNVs), and 153 were deleterious VUS. Patients with deleterious VUS did not have different survival compared with those with no variants, and these were not considered for designating unrecognized IBMFS and SPVR casesOur analysis showed that 6.7% (N=49/732) of patients had variants consistent with an unrecognized IBMFS; 22 were AR (21 compound heterozygous, 1 homozygous), 26 AD and 1 XLR. Approximately half of those patients (22/49, 45%) had P/LP variants in hematopoiesis genes, and 31% in telomere biology genes (Figure 1A). We identified 79 patients with an SPVR, most in SBDS and FANCM.Patients with an unrecognized IBMFS had worse overall survival when compared with patients with presumed acquired SAA (log-rank p=0.0098) (Figure 1B). Multivariable analysis confirmed this association (HR=2.11, 95% confidence interval (CI)=1.38-3.22, p=0.001). The observed survival difference was not mitigated by lower conditioning regimen intensity (HR=2.1, p=0.01 in myeloablative condition (MAC), and HR=2.3, p=0.016, in reduced intensity regimens (RIC)).Patients with an SPVR had no post-HCT survival difference than presumed acquired SAA regardless of conditioning regimen (overall HR=0.96, p=0.85; MAC HR=1.4, p=0.27; RIC HR=0.3, p=0.1).Conclusions: A sizable subset of patients (6.7%) with reported immune mediated acquired SAA had unrecognized inherited disorder and 33% were adults at HCT. Unrecognized IBMFS was associated with statistically significant poorer survival after HCT. In contrast, post-HCT survival in patients with an SPVR (“carriers”) was not different than those with acquired SAA. This work underscores the importance of identifying SAA patients with clinically meaningful underlying inherited disorders to enable the use therapeutic approaches to minimize regimen toxicity and late complications. It further highlights that identification of a single pathogenic variant in an autosomal recessive gene or X-linked gene in females and VUS are associated with post-HCT survival similar to acquired SAA. [Display omitted] DisclosuresPaczesny: Medical University of South Carolina: Patents & Royalties: inventor on the ST2 bispecific antibody patent application. Lee: Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; JANSSEN: Other; AstraZeneca: Research Funding; Takeda: Research Funding; Novartis: Other: clinical trials, Research Funding; Syndax: Research Funding; Pfizer: Research Funding; Incyte: Research Funding.

Repeated injury promotes tracheobronchial tissue stem cell attrition.

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue‐specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide‐label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long‐ and short‐lived TSC and showed that short‐lived TSC clones had significantly shorter telomeres than their long‐lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation.

Evaluating The Utility Of Telomere Length Measurement By Qpcr As a Diagnostic Test For Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (IBMFS) classically diagnosed by the triad of dysplastic nails, reticular skin pigmentation, and oral leukoplakia. Patients with DC are at very high risk of bone marrow failure (BMF), cancer, pulmonary fibrosis, and other medical problems. Germline mutations in key telomere biology genes cause DC (DKC1, TERC, TERT, TINF2, NOP10, NHP2, WRAP53, CTC1 or RTEL1), although about 30% of patients lack a known mutation. Telomere length (TL) below the1st percentile for age in leukocyte subset, measured by flow cytometry with in situhybridization (flow FISH), is diagnostic of DC with a sensitivity and specificity of more than 95%. However, flow FISH requires fresh or cryopreserved blood, and thus is relatively low-throughput and costly. Quantitative PCR (QPCR) of telomeric DNA content, expressed as the ratio of the telomeric product (T) to a single copy gene (S) product, is a high-throughput, inexpensive method of determining relative TL (RTL) that is being used in numerous studies.We evaluated the clinical utility of QPCR RTL measurement as a diagnostic test for DC in the NCI's IRB-approved longitudinal cohort study of IBMFS. Patients were classified as DC (n=31) if they had a mutation in one of the nine known causative genes and/or met the clinical criteria for DC including at least two features of the diagnostic triad with other clinical findings consistent with DC. Fifty-one mutation-negative, healthy relatives of DC patients served as controls. Flow FISH lymphocyte TL was less than or equal to the 1st percentile for age in 29/31 (94%) DC patients and above 1stpercentile in all but one mutation-negative relative. Monoplex QPCR of DNA derived from whole blood was used to determine the RTL (T/S ratio). QPCR RTL data were log transformed and standardized for age using percentile curves and z-scores calculated from 477 healthy age-matched controls. Control samples from children were derived from the control arm of the eating behavior and obesity studies at the National Institute of Child Health and Human Development, NIH, and adult samples were from healthy donors in the National Marrow Donor Program research sample repository. We used Mann-Whitney and Kruskal-Wallis tests to compare z-scores of TL between categories and Receiver Operating Curve (ROC) analysis to calculate the sensitivity and specificity of QPCR RTL at different percentile cut-offs.DC patients had significantly shorter QPCR RTL than relatives (median age adjusted z-score -1.9 vs. 0.09, p<0.0001). Patients with a mutation in TINF2, or DKC1had shorter QPCR relative TL than those with other mutations (p=0.07). DC patients who had at least two of the three features of the diagnostic triad had shorter QPCR relative TL than those who had one or zero features (p=0.05), but there was no difference in QPCR TL between individuals based on severity of bone marrow failure (p=0.2).QPCR RTL was not correlated with flow FISH TL in DC patients (r=0.09, p=0.64). QPCR RTL was only modestly correlated with flow FISH TL in unaffected relatives (r=0.47, p=0.001). QPCR RTL less than the 1st percentile for age missed diagnosing 60% of the known DC patients as having DC (sensitivity=38.7%, and specificity=98%). Only 75% of DC patients had QPCR relative TL below the 10thpercentile for age (sensitivity=74%), as did 12% of the unaffected relatives (specificity=88%).In summary, QPCR RTL measurement less than 1st percentile for age had low sensitivity for identifying patients with DC. Raising the cutoff to the 10th percentile increased the sensitivity, but 12% of healthy individuals were also falsely diagnosed with DC. Our findings raise doubt about using QPCR for clinical diagnosis of DC. This has implications in the interpretation of studies utilizing different TL measurement methods in studies of DC, bone marrow failure, and related telomere biology disorders. With the current limitations of QPCR RTL in identifying DC patients, 6-panel flow FISH TL continues to be the recommended diagnostic test for DC. Disclosures:No relevant conflicts of interest to declare.

Disease Progression and Outcomes in Patients with Telomere Biology Disorders

Dyskeratosis congenita and related telomere biology disorders (DC/TBDs) are multisystem illnesses caused by germline pathogenic variants (PV) in telomere biology genes resulting in very short telomeres. The inheritance varies and includes autosomal dominant (AD), autosomal recessive (AR) and X-linked (XLR) disease. Young patients may present with bone marrow failure (BMF) and the mucocutaneous triad of dysplastic nails, abnormal skin pigmentation, and oral leukoplakia, whereas adults may be identified with isolated BMF, pulmonary fibrosis (PF), or liver disease. In addition to BMF, DC/TBDs are associated with high risk of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), head and neck squamous cell cancer (HNSCC) and many other medical problems. Hematopoietic cell transplant (HCT), lung, and/or liver transplantation may be required. A limited number of genotype-phenotype studies suggest the mode of inheritance to be associated with phenotypes. We studied 229 individuals (144 males, 85 females) with DC/TBDs enrolled in an IRB-approved longitudinal cohort study of inherited BMF syndromes (NCT00027274) between 01/2002 and 05/2019. Questionnaires and medical records were reviewed and a subset of 93 patients (40.6%) was systematically evaluated at the NIH Clinical Center (CC) during a 5-day visit. The median age at diagnosis for the entire cohort was 23.4 (0-82.2) years and median follow-up time was 4.2 (0-36.2) years. The PV was identified in 180 (78.6%) patients: 98 had AD disease (TERT, TERC, RTEL1, ACD, or PARN), 58 had XLR or AR (TERT, RTEL1, ACD, WRAP53, PARN, or DKC1) and 24 TINF2 (AD or de novo). In the entire cohort of 229 patients, 69 (30.1%) required HCT for BMF [median age 14.9 (0.9-63.1) years] and 58% of these had AR/XLR or TINF2-associated disease. Eight patients received lung transplant for PF [median age 51.4 (13.1-62.4) years]. Liver transplant was required in 5 patients [median age 25.8 (9.9-56.7) years]. Twenty-seven non-HCT patients developed cancer [median age of 42.1 (18.2-67.5) years]. In 5 patients, cancer occurred after HCT [median time since HCT=4.6 (1.8-14.6) years] and 3 developed post-HCT lymphoproliferative disease. Ninety-three patients were systematically phenotyped at the CC; 52 (55.9%) had &amp;gt;=1 triad feature and 33 (35.5%) had severe BMF. Three non-HCT patients had a history of cancer. PF was present in 6 patients and 3 had severe liver disease (portal hypertension and/or liver cirrhosis/fibrosis). Esophageal strictures were present in 7 and 5 had hip avascular necrosis (AVN, non-HCT). Median follow-up time after CC visit was 7.1 (0-17.1) years with median age at last follow-up of 29.6 (2.2-79.5) years . During follow-up, 9 patients progressed to severe BMF, and 8 developed cancer (4 post-HCT). Ten developed PF (5 post-HCT), and 9 advanced to pulmonary arteriovenous malformations (8 post-HCT). Eight patients progressed to severe liver disease (7 post-HCT). Gastrointestinal telangiectasias were seen in 6 individuals. Four patients developed esophageal stricture and 10 AVN (7 after radiotherapy and/or HCT). At last follow up of the entire cohort, 92 (40.2%) of 229 patients were deceased. The main causes of death were lung disease (28.3%), treatment-related complications (19.6%), cancer (16.3%), or BMF (14.1%). The median overall survival was 54.2 years [95% confidence interval (CI) 47.3-59.8] and HCT-free median survival was 46.9 years (95% CI 37.9-54.2). Overall survival was significantly better in patients with AD versus XLR/AR or TINF2 disease (p&amp;lt;0.0001, log-rank test, median survival 66.5 versus 33.2 years, respectively). Our study systematically quantified the presence and progression of DC/TBDs clinical manifestations and illustrates the significant morbidity faced by patients with these multisystem disorders. Long term survival is poor overall, but significantly better in patients with AD disease compared with other inheritance patterns. Although HCT has improved outcomes related to BMF, this study illustrates the pressing need to develop therapies for patients with DC/TBDs. These data also form the foundation for incorporating the mode of inheritance into evidence-based clinical care guidelines, risk stratification, and possibly treatment decisions in patients with DC/TBDs. Disclosures No relevant conflicts of interest to declare.

A Mendelian randomization study of telomere length and blood-cell traits

Whether telomere attrition reducing proliferative reserve in blood-cell progenitors is causal has important public-health implications. Mendelian randomization (MR) is an analytic technique using germline genetic variants as instrumental variables. If certain assumptions are met, estimates from MR should be free from most environmental sources of confounding and reverse causation. Here, two-sample MR is performed to test whether longer telomeres cause changes to hematological traits. Summary statistics for genetic variants strongly associated with telomere length were extracted from a genome-wide association (GWA) study for telomere length in individuals of European ancestry (n = 9190) and from GWA studies of blood-cell traits, also in those of European ancestry (n ~ 173,000 participants). A standard deviation increase in genetically influenced telomere length increased red blood cell and white blood cell counts, decreased mean corpuscular hemoglobinand mean cell volume, and had no observable impact on mean corpuscular hemoglobin concentration, red cell distribution width, hematocrit, or hemoglobin. Sensitivity tests for pleiotropic distortion were mostly inconsistent with glaring violations to the MR assumptions. Similar to germline mutations in telomere biology genes leading to bone-marrow failure, these data provide evidence that genetically influenced common variation in telomere length impacts hematologic traits in the population.

Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita

AbstractDyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3′ region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3′-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3′ to 5′ degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.

Germinal Predisposition in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction: Identification of Deleterious Variants in Microsatellite Instability Genes

Germinal Predisposition in Myelodysplastic Syndromes in Young Adults without a Preexisting Disorder or Organ Dysfunction: Identification of Deleterious Variants in Microsatellite Instability Genes

Telomere Elongation and Clinical Improvement in Telomeropathy Patients: A Prospective Clinical Trial of Nandrolone in Telomeropathies

Telomeres are a complex of hexameric repetitive DNA sequences at the end of linear chromosomes. Telomeres erode during mitosis, and this process is accelerated in patients carrying germline pathogenic mutations in telomere-biology genes (Calado and Young, NEJM 2009). In vitro, the exposition to androgens stimulates TERT transcription and telomerase activity of lymphocytes and bone marrow CD34+ cells (Calado et al. Blood 2009). In a phase 1-2 prospective trial, the use of danazol led to telomere elongation in patients with telomeropathies (Townsley et al. NEJM 2016). The hematological response was observed in 79% of evaluable patients. Here, we describe a phase 1-2 single-center prospective study assessing the safety and the effect of the male hormone nandrolone decanoate on telomere erosion in patients with telomere diseases (ClinicalTrials.gov Identifier: NCT02055456). Entry criteria comprised patients older than two-year-old with age-adjusted mean telomere length below the 1st percentile ± identified mutations in telomerase complex genes, associated with at least one cytopenia and/or diagnosis of idiopathic pulmonary fibrosis (IPF). Patients were treated with nandrolone decanoate intramuscular at a dose of 5 mg/kg every 14 days for 24 months. The primary efficacy end point was a 20% decrease in the annual rate of telomere erosion, and the incidence of toxic effects was the primary safety end point. Secondary endpoints were hematologic response and pulmonary response. Flow-FISH was used to determine the telomere length of peripheral blood leukocytes. From May 2014 to October 2017, 20 consecutive patients were eligible for participation, and 17 were enrolled. The median age was 36 years (range, 4 - 59 years), and five patients were female. In all but one patient germline pathogenic mutations were identified (TERT, 7; RTEL1, 4; TERC, 2; WRAP53, 1; TINF2, 1; and SAMD9, 1). Eleven patients were diagnosed with bone marrow failure (9 with moderate and one with severe AA, and one with myelodysplasia). Four patients were diagnosed with IPF, and 2 patients had both moderate AA and IPF. One patient diagnosed with IPF had received an allogeneic bone marrow transplant before enrollment. Four patients had additional hepatic involvement, and 5 displayed cutaneous features of dyskeratosis congenita. The most common adverse events associated with drug were elevations in liver enzyme levels in 88%, acne in 59%, and virilization in 59%. Severe adverse events possibly related to drug occurred in six instances. Dose reductions were necessary in 5 patients due to severe or moderate adverse events. Seven patients withdrew from the study before 2 years: two patients discontinued therapy due to grade 3 adverse events (depression and acne); one patient sought alternative therapies; and four patients died during the study period, two due progressive IPF and two due intracranial hemorrhage. Out the 17 patients enrolled, a total of 15 reached 12 months of treatment and 13 were evaluable (one patient was transplanted and telomere length not evaluated; and for one patient the telomere sample was missing). Ten patients met the primary efficacy end point, showing telomere elongation. As of July 2019, nine patients reached 24 months of treatment and telomere length measured for seven patients at this time-point; all of them met end point criteria and showed telomere elongation at two years. The average increase in telomere length at 12 months was 1,119 bp (95% CI, 180-2,059 bp; P&lt;0.04), and 1,257 bp (95% CI, 675-1,839 bp; P&lt;0.02) at 24 months. The hematologic response rate was 60% at 3 months and 80% at 6 months. Only one patient relapsed during nandrolone use. Five of 8 transfusion-dependent patients became transfusion-independent, and one patient showed a reduction in transfusion requirements &gt;50%. The pulmonary response rate was observed in 2 of 6 patients who had a pulmonary disease at baseline, and one patient eventually became oxygen-independent. In our study, nandrolone decanoate treatment was safe and led to telomere elongation in patients with telomeropathies. Nandrolone also improved hematologic function in patients with marrow failure. Pulmonary function remained stable or ameliorated in two-thirds of patients with pulmonary involvement. Known nandrolone side effects were common, especially liver toxicity and virilization. OffLabel Disclosure: We want to determine if treatment with male hormone nandrolone decante is safe and improves hematologic response as first-line treatment in patients with telomere disease.

A novel homozygous RTEL1 variant in a consanguineous Lebanese family: phenotypic heterogeneity and disease anticipation.

Phenotypic heterogeneity is often observed in patients with telomeropathies caused by pathogenic variants in telomere biology genes. However, the roles of recessive variants in these different phenotypes are not fully characterized. Our goal is to describe the biological roles of a novel homozygous RTEL1 variant identified in a consanguineous Lebanese family with unusual presentation of telomeropathies. A proband was screened for germline variants in telomere biology genes by whole exome sequencing. Leukocytes' telomere length was measured in the proband and eight relatives. We identified a novel homozygous p.E665K RTEL1 variant in the proband, his mother, and seven siblings that associated with telomere shortening and a broad spectrum of clinical manifestations, ranging from mild unspecific findings to severe phenotypes. Consanguinity in at least three family generations led to increased frequency of the homozygous p.E665K variant in the youngest generation and progressive telomere shortening. The increased frequency of the homozygous RTEL1 variant due to consanguinity in this Lebanese family allowed us to infer novel behaviors of recessive RTEL1 variants, as the expressivity and penetrance of this gene are very heterogenous between inter- and intra-generations. Progressive telomere shortening was associated with disease anticipation, first reported in recessive autosomal telomeropathies. Both genetic testing and telomere length measurement were critical for the clinical diagnosis of this family with telomere diseases marked by phenotypic heterogeneity.

Pathogenic TERT promoter variants in telomere diseases

PurposeThe acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes. MethodsWe screened patients with a broad spectrum of telomeropathies (n=136), their relatives (n=52), and controls (n=195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay. ResultsPathogenic −124 and −146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging. ConclusionWe extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.

HiChIRP reveals RNA-associated chromosome conformation.

Modular domains of long non-coding RNAs can serve as scaffolds to bring distant regions of the linear genome into spatial proximity. Here, we present HiChIRP, a method leveraging bio-orthogonal chemistry and optimized chromosome conformation capture conditions, which enables interrogation of chromatin architecture focused around a specific RNA of interest down to approximately ten copies per cell. HiChIRP of three nuclear RNAs reveals insights into promoter interactions (7SK), telomere biology (telomerase RNA component) and inflammatory gene regulation (lincRNA-EPS).

Somatic TERT promoter Mutations in a Broad Spectrum of Telomeropathies

Telomerase reactivation by acquisition of mutations in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. The most common TERTp mutations are located in positions -146, -124, and -57 upstream the initiation codon. In non-malignant diseases, TERTp mutations only have been reported in patients with idiopathic pulmonary fibrosis (IPF) caused by germline mutations in telomere biology genes, that are also etiologic in a broader spectrum of diseases collectively named telomeropathies (such as IPF, aplastic anemia [AA], dyskeratosis congenita [DC], and cirrhosis).We screened blood from 136 patients with telomeropathies (median age=29 years; range, 1-76), 52 relatives (median age=40 years; range, 8-72), and 195 controls using a customized low-cost amplicon-based next-generation sequencing (NGS) assay for identification and quantification TERTp mutations. Patients had DC (n=21), AA (n=86), IPF with or without another telomeropathy-related phenotype (n=18), or other phenotypes (n=11). Inclusion criteria were telomere length (TL) below the 10th percentile of age-matched controls or a germline mutation in a telomere-related gene classified as pathogenic/likely pathogenic or of uncertain significance by the ACMG criteria. Patients' relatives were only studied if they carried the same germline mutation as the proband or had short telomeres, regardless of symptoms or evidence of disease. Patients with acquired AA (n=70), IPF (n=12), other inherited bone marrow failure (n=7), and acute myeloid leukemia (AML; n=106) were controls. All TERTp mutations identified by NGS were confirmed and tracked over time by droplet digital PCR.We identified the -124 or -146 mutations in leukocytes from 12 unrelated patients diagnosed with IPF (n=6), DC (n=2), or moderate AA (n=4). Five relatives also had the -146 (n=1), and -124 (n=4) mutations, all carriers of a germline mutation in telomere biology gene. The frequency of TERTp mutations was much higher in IPF patients compared to AA cases (33% vs. 4.6%; Fisher's exact test, P=0.0016). However, no difference in frequency of TERTp mutations among patients with IPF vs. marrow failure was observed (41% vs. 58%; Fisher's exact test, P>0.05), suggesting TERTp mutations occurred in both clinical presentations. MutTERTp clones positively correlated with age, as they were only present in individuals older than 18 years old and more frequent in those 60 to 80 years old. Also, TERTp mutations more frequently co-ocurred with germline TERT mutations (n=13) compared to mutations in TERC (n=2), RTEL1 (n=1), or DKC1 (n=2) (76% vs. 23%; Fisher's exact test, P=0.002). All germline variants were pathogenic or had some evidence of pathogenicity. MutTERTp clones size varied from 1.2% to 50% in total leukocytes and was at higher allele frequencies (VAF) in the granulocytic fraction from four patients. In serial samples (available for five patients), the mutTERTp clone size expanded over time, suggesting a selective growth advantage in comparison to unmutated hematopoietic cells. Despite that, mutTERTp clones did not associate with blood counts or telomere elongation; most subjects carrying a TERTp mutation, which is known to upregulate TERT expression, nevertheless had short or very short telomeres (15 out of 17 individuals). Six patients with mutTERTp clones (VAF ranging from 3-33% in myeloid cells) were treated with danazol for two years; four were responders and two were off-study after 3-6 months. In serial samples (available for two patients), the mutTERTp clone sizes decreased during danazol treatment while blood counts improved. After treatment, mutTERTp clones VAF increased. TERTp mutations were found in telomeropathy patients who had a germline variant in telomere biology genes but not in controls or patients with very short telomeres without germline variant in telomere biology genes.We have expanded the spectrum of non-malignant diseases associated with somatic TERTp mutations to DC, AA, and cirrhosis. Our data indicate that mutTERTp clones are specifically and randomly selected with aging in a marrow environment deficient in telomerase function, and mutTERTp selection did not associate with patients' peripheral blood counts, TL, and response to danazol treatment. TERTp emergence may be a useful clonal indicator for telomere dysfunction and may help to assess the pathogenicity of unclear constitutional variants in telomeropathies. DisclosuresYoung:CRADA with Novartis: Research Funding; National Institute of Health: Research Funding; GlaxoSmithKline: Research Funding.

Beginning at the ends: telomeres and human disease.

Studies of rare and common illnesses have led to remarkable progress in the understanding of the role of telomeres (nucleoprotein complexes at chromosome ends essential for chromosomal integrity) in human disease. Telomere biology disorders encompass a growing spectrum of conditions caused by rare pathogenic germline variants in genes encoding essential aspects of telomere function. Dyskeratosis congenita, a disorder at the severe end of this spectrum, typically presents in childhood with the classic triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia, accompanied by a very high risk of bone marrow failure, cancer, pulmonary fibrosis, and other medical problems. In contrast, the less severe end of the telomere biology disorder spectrum consists of middle-age or older adults with just one feature typically seen in dyskeratosis congenita, such as pulmonary fibrosis or bone marrow failure. In the common disease realm, large-scale molecular epidemiology studies have discovered novel associations between illnesses, such as cancer, heart disease, and mental health, and both telomere length and common genetic variants in telomere biology genes. This review highlights recent findings of telomere biology in human disease from both the rare and common disease perspectives. Multi-disciplinary collaborations between clinicians, basic scientists, and epidemiologist are essential as we seek to incorporate new telomere biology discoveries to improve health outcomes.