Telomere Biology Research Articles

Impact of Dkc1 Deficiency on BM Niche Function during Hematopoietic Stem Cell Transplantation in a Conditional Murine Model of X-Linked Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a telomere biology disorder associated with bone marrow failure (BMF) and the triad of dystrophic nails, skin rashes, and leukoplakia. X-linked DC (XDC) is caused by mutations in DKC1, which encodes dyskerin, a nucleolar protein associated with rRNA processing and telomerase complex formation. Hematopoietic stem cell transplantation (HSCT) is currently the only definitive curative treatment for BMF in XDC patients, though many patients exhibit only partial restoration of hematopoietic function. Our previous studies demonstrated that molecular and cellular defects in BM niches contribute to poor donor HSC engraftment in murine models of other BMF syndromes, including Shwachman-Diamond Syndrome (SDS), a disease whose pathogenesis intersects with the role of dyskerin in ribosome biogenesis. These studies led us to hypothesize that BM niche dysfunction contribute to poor graft function in DC patients post-HSCT. Defining specific deficits would enable development of novel targeting strategies to improve HSCT outcomes for patients with XDC. Here, we report that in a novel murine model of XDC driven by conditional Dkc1 deletion, Dkc1 deficiency within mesenchymal BM niche cells causes significant impairment of healthy donor HSC engraftment after HSCT. We have identified defects in multiple pathways within Dkc1-deficient BM niche in response to myeloablative conditioning that may drive this reduction in HSC supportive capacity. To generate a murine model with conditional Dkc1 deletion in BM niche, we crossed Dkc1l/lmice with Mx1 Cre+mice, inducing Cre expression in hematopoietic cells and Mx1-inducible niche cells by polyinosinic-polycytidylic acid administration. At baseline, the resulting Mx1 CreDkc1 Exc mice exhibit mild hematopoietic abnormalities versus Dkc1 l/l controls, including variable thrombocytopenia, increased blood monocytes, and reduced BM lymphoid progenitors. To assess capacity of BM niches from Mx1 CreDkc1 Exc mice to efficiently engraft donor HSC, we transplanted Mx1 CreDkc1 Exc mice and Dkc1 l/l controls with healthy GFP + wild-type donor BM and assessed efficiency of donor HSC engraftment at 2 weeks following HSCT using competitive secondary HSCT. The Mx1 CreDkc1 Exc recipients exhibited significantly impaired efficiency of donor HSC engraftment compared with controls, as indicated by a 25-30% reduction of long-term GFP + cell reconstitution in mature blood lineages in secondary recipients (Figure 1). We next sought to identify molecular and cellular defects in BM niche that contribute to the engraftment deficits in Mx1 CreDkc1 Exc mice. Flow cytometry analysis of BM niche cells 48 hours after total body irradiation (TBI) showed Mx1 CreDkc1 Exc mice exhibited a 20% reduction of CD11b+F4/80+ osteomacs compared to Dkc1 l/l controls. In contrast to our published findings in a model of SDS, the frequencies of osteoprogenitors, mesenchymal stem cell, endothelial cells and megakaryocytes in BM niche were similar between irradiated Dkc1-deficient and control mice. We then performed multiplex ELISA and RNA-seq analyses on isolated BM niche cells from Mx1 CreDkc1 Exc versus control BM at 48 hours post-TBI. Irradiated Mx1 CreDkc1 Exc BM niche demonstrated lower expression of multiple chemokines, including CXCL1, CXCL12, CCL1, CCL11 and CCL12, known to be critical for recruiting HSC and many leukocyte subsets. Dkc1 deficiency in post-TBI BM niche cells also led to lower expression of genes that regulate production of IFN-γ and IL-1, resulting in reduced expression level of both cytokines that may drive decreased osteomacs seen in Dkc1-deficient BM niches. Moreover, Gene Ontology analysis indicates that after TBI, Dkc1 deficiency within BM niches resulted in downregulation of gene expression involved in cell-cell adhesion and extracellular matrix, both of which are critical for HSC enlodgement and engraftment after HSCT. Taken together, our results demonstrated that Dkc1 deficiency in BM niche cells that are not replaced by HSCT impairs the ability of these niches to engraft donor HSC efficiently. Disruptions of molecular and cellular pathways implicated in the niche dysfunction of this XDC model are distinct from our previous findings with SDS model. Ongoing and future studies are needed to define which of these pathways have the highest potential to serve as translational targets to improve HSCT success rate to treat BMF in patients with XDC.

Blood Count Recovery and Telomere Elongation in a TINF2 Patient Under Treatment with a Gonadotropin-Releasing Hormone (GnRH) Agonist

Telomere biology disorders (TBDs) caused by germline variants in TINF2 are a common cause of severe disease presenting in early childhood. Progressive bone marrow failure in patients with TBDs is often treated with androgens, known to improve hematopoiesis but with a controversial impact on telomere elongation. Here, we report a unique case of a young TINF2 male patient that had a striking recovery of blood counts and robust telomere elongation in peripheral blood cells during treatment with histrelin, a gonadotropin-releasing hormone (GnRH) agonist and inhibitor of testosterone production. We investigated whether underlying clonal events were linked to blood counts and telomere length (TL) dynamics. We extracted medical information and collected peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) over an 10-yr period beginning at diagnosis (age 4), which was based on cytopenias, marked marrow hypocellularity, very low TL in all leukocyte subsets by Flow-FISH, and a de novo TINF2 p.K302Rfs*17 germline variant. Following 2 yrs of observation, he was started on danazol for progressive cytopenias but it was discontinued after 21 months due to the onset of danazol-related precocious puberty, for which histrelin was administered via subcutaneous implants over 4.7 yrs. Hemoglobin levels normalized while on danazol and remained stable at last follow-up. Although platelet counts increased and stabilized at ~ 40,000 on danazol, they steadily increased after initiation of histrelin up to 156,000; they then gradually declined to 108,000 over the 18 months following removal of the last histrelin implant (Fig. 1A). The absolute neutrophil count normalized while on histrelin and has remained in normal range. While TLs remained very low in all leukocyte subsets while on danazol, they either stabilized or minimally changed (+/- 0.2 kb) in lymphocytes, CD45RA+, CD45RA-, and CD57+ cells or increased by 2.8 kb or 1 kb in granulocytes and B cells, respectively. While on histrelin, TL further increased in the lymphocytes, granulocytes, and B cells to max gains of 0.4 kb, 3 kb, and 2 kb, respectively. Fourteen months after discontinuing histrelin, TL shortened in all subsets. Screening of PBMC at 8.5 yrs of follow-up for clonal hematopoiesis (CH) in myeloid and telomere-related genes by error-corrected DNA sequencing identified POT1 p.D291Y and TINF2 p.W20* variants at variant allele frequencies (VAFs) of 20% and 4%, respectively, as likely somatic genetic rescue (SGR) mechanisms (Fig. 1B). POT1 D291Ywas retrospectively tracked at a VAF of 0.7% in total DNA before any treatment and found at a VAF of 8% in PBMC collected during danazol treatment, 27% during histrelin treatment, and 14% after histrelin discontinuation. TINF2 W20* emerged while on histrelin at a VAF of 1% in PBMC; this mutation VAF slightly increased during histrelin treatment but robust clonal expansion up to a VAF of 21% was not seen until off histrelin. Hematologic improvement, particularly thrombocytopenia, and continued telomere elongation in granulocytes and B cells (CD20+) coincided with clonal expansion of the POT1 D291Y variant. Single-cell proteogenomic sequencing of BMMNCs evidenced POT1 D291Y and TINF2 W20* as independent clonal events that emerged in CD34+ hematopoietic stem cells (HSC) and were present both in the megakaryocytic and erythroid, and myeloid and lymphoid progenitor cells. During histrelin treatment, POT1 D291Y dominated the landscape and associated with increased lymphocyte counts, being present in 30% of HSC and progenitor cells, myeloid cells, and T lymphocytes. When histrelin was discontinued, hematopoiesis greatly shifted toward myeloid production, which coincided with TINF2 W20*clonal expansion and a substantial reduction of progenitors and CD8+ cells. Single-cell RNAseq of BM HSC revealed similar transcriptional profiles while on danazol and histrelin compared to baseline, with up and downregulation of inflammatory pathways and MYC targets, respectively; these programs were enhanced while on histrelin compared to danazol. In summary, we characterized a unique combination of SGR events in a patient with a TINF2 germline mutation, which can guide future research on new therapeutic strategies. As opposed to androgens, histrelin promoted the specific expansion of POT1 clones with proliferation of HSC and progenitor cells, improved hematopoiesis, and increased TL.

Polygenic Modifiers of Expressivity in Telomere Biology Disorders

Telomere biology disorders (TBDs) are caused by pathogenic germline variants in genes involved in telomere maintenance resulting in very short telomeres for age and high risks of bone marrow failure, certain cancers, as well as pulmonary and liver disease. Interestingly, some pathogenic TBD mutations have variable expressivity and incomplete penetrance, even within families. Variant carriers can present with early manifestations such as the mucocutaneous triad and bone marrow failure or may not manifest until later in life with adult-onset idiopathic pulmonary fibrosis or liver disease, or have no associated clinical phenotype. The underlying causes of this heterogeneity are poorly understood. We hypothesized that common genetic variants contributing to inter-individual variation in telomere length in the general population may combine with the effects of monogenic TBD-causing variants to underlie variable clinical manifestations in TBDs. To examine this, we developed polygenic scores (PGS) to predict telomere length using genome-wide SNPs from the UK Biobank. We then applied the most predictive polygenic score to the out-of-sample All of Us cohort, as well as the National Cancer Institute's longitudinal cohort of individuals with inherited bone marrow failure syndromes, including those with TBDs (ClinicalTrials.gov Identifier: NCT00027274). If monogenic mutations and polygenic predisposition to short telomeres both contribute to the clinical severity of TBDs and likelihood of early-onset manifestations, the distribution of genetically predicted telomere length in this ascertained cohort would be shifted towards shorter telomeres compared with the population average. Consistent with this, individuals with TBDs enriched for early-onset bone marrow failure phenotypes (n = 92) have a median polygenic score 0.32 standard deviations (SDs) shorter than the UK Biobank (p=0.002), and 0.31 SDs shorter than the external All of Us cohort (p=0.003) ( Figure). Individuals with TBDs also have shorter genetically predicted telomeres compared to individuals with Fanconi anemia (n = 45) and Diamond Blackfan anemia (n = 49) (p=0.008), indicating that this phenomenon is specific to TBDs. We binned the PGS into quintiles and show that the lowest quintile is associated with an odds ratio of TBD of 1.89 [1.19,2.86], while being in the highest quintile is associated with an odds ratio of 0.54 [0.29,1.02]. Interestingly, TBD cases with no identified mutation have a median PGS 0.1 SDs towards shorter telomeres than patients with a known mutation. Collectively, these results indicate that common, genome-wide determinants of telomere length are significant modifiers of the early-onset manifestations of TBDs. We hypothesized that the converse should also be true: TBD causal variants should be present in adult population cohorts, and adult individuals with a pathogenic variant who avoided the severe childhood-onset manifestations of TBDs, should not have polygenically predicted short telomere length. To test this hypothesis, we filtered the UK Biobank for TBD causal variants annotated in ClinVar, and identified 71 putative pathogenic cases. Importantly, among these cases there were no individuals with bone marrow failure, but these individuals did have shorter telomeres compared with the UK Biobank. Interestingly, these 71 putative cases had a population-normal telomere length PGS, and significantly longer PGS predicted telomere length than the NCI cohort (p=0.025). We also find that being classified as among these pathogenic cases is associated with an odds ratio for developing pulmonary fibrosis of 10.79 [4.34,26.8]. In summary, despite normal PGS and absence of bone marrow failure, these putative cases have short telomeres and increased risk of adult manifestations of TBDs, consistent with the idea that the pathogenic mutations and common genetic variation affecting telomere length combine to impact expressivity. Collectively, these findings indicate that polygenic effects on telomere length can modify the impacts of monogenic mutations in TBD-associated genes. These results have implications not only for clinical risk assessment in patients with TBDs, but also for our general understanding and interpretation of variable penetrance/expressivity in presumed monogenic disorders.

A novel telomere biology disease-associated gastritis identified through a whole exome sequencing-driven approach.

A whole exome sequencing (WES)-driven approach to uncover the etiology of unexplained inflammatory gastritides has been underutilized by surgical pathologists. Here, we discovered the pathobiology of an unusual chronic atrophic gastritis in two unrelated patients using this approach. The gastric biopsies were notable for an unusual pattern of gastritis with persistent dense inflammation, loss of both parietal and neuroendocrine cells in the oxyntic mucosa, and sparing of the antral mucosa. The patients were found to harbor pathogenic variants in telomeropathic genes (POT1 and DCLRE1B). Clonality testing for one of the patients showed evidence of evolving clonality of TCR-gene rearrangement. Both patients showed significantly decreased numbers of stem/progenitor cells by immunohistochemistry, which appears to be responsible for the development of mucosal atrophy. No such cases of unusual chronic atrophic gastritis in the setting of telomeropathy have been previously reported. The loss of stem/progenitor cells suggests that stem/progenitor cell exhaustion in the setting of telomere dysfunction is the likely mechanism for development of this unusual chronic atrophic gastritis. The results underscore the need for close monitoring of these gastric lesions, with special regard to their neoplastic potential. This combined WES-driven approach has promise to identify the cause and mechanism of other uncharacterized gastrointestinal inflammatory disorders.

Implementation of a prospective screening strategy to identify adults with a telomere biology disorder among those undergoing lung transplant evaluation for interstitial lung disease

IntroductionPatients with interstitial lung disease (ILD) secondary to telomere biology disorders (TBD) experience increased morbidity after lung transplantation. Identifying patients with TBD may allow for personalized management to facilitate better outcomes. However, establishing a TBD diagnosis in adults is challenging. MethodsA TBD screening questionnaire was introduced prospectively into the lung transplant evaluation. Patients with ILD screening positive were referred for comprehensive TBD phenotyping and concurrent telomere length measurement and germline genetic testing. ResultsOf 98 patients, 32 (33%) screened positive. Eight patients (8% of total; 25% of patients with a positive screen) met strict TBD diagnostic criteria, requiring either critically short lymphocyte telomeres (<1st percentile) (n = 4), a pathogenic variant in a TBD-associated gene (n = 1), or both (n = 3) along with a TBD clinical phenotype. Additional patients not meeting strict diagnostic criteria had histories consistent with TBD along with telomere lengths <10th percentile and/or rare variants in TBD-associated genes, highlighting a critical need to refine TBD diagnostic criteria for this patient population. ConclusionA TBD phenotype screening questionnaire in patients with ILD undergoing lung transplant evaluation has a diagnostic yield of 25%. Additional gene discovery, rare variant functional testing, and refined TBD diagnostic criteria are needed to realize the maximum benefit of testing for TBD in patients undergoing lung transplantation.

Telomere biology disorders may manifest as common variable immunodeficiency (CVID)

Telomere biology disorders (TBD) are caused by germline pathogenic variants in genes related to telomere maintenance and are characterized by critically short telomeres. In contrast to classical dyskeratosis congenita (DC), which is typically diagnosed in infancy, adult or late onset TBD frequently lack the typical DC triad and rather show variable organ manifestations and a cryptic disease course, thus complicating its diagnosis. Common variable immunodeficiency (CVID), on the other hand, is a primary antibody deficiency (PAD) syndrome. PADs are a heterogenous group of diseases characterized by hypogammaglobulinemia which occurs due to dysfunctional B lymphocytes and additional autoimmune and autoinflammatory complications. Genetic screening reveals a monogenic cause in a subset of CVID patients (15–35%). In our study, we screened the exomes of 491 CVID patients for the occurrence of TBD-related variants in 13 genes encoding for telomere/telomerase-associated proteins, which had previously been linked to the disease. We found 110/491 patients (22%) carrying 91 rare candidate variants in these 13 genes. Following the American College of Medical Genetics and Genomics (ACMG) guidelines, we classified two variants as benign, two as likely benign, 64 as variants of uncertain significance (VUS), four as likely pathogenic, and one heterozygous variant in an autosomal recessive disease gene as pathogenic. We performed telomere length measurement in 42 of the 110 patients with candidate variants and CVID. Two of these 42 patients showed significantly shorter telomeres compared to controls in both lymphocytes and granulocytes. Following the evaluation of the published literature and the patient's manifestations, we re-classified two VUS as likely pathogenic variants. Thus, 0.5–1% of all CVID patients in our study carry possibly pathogenic variants in telomere/telomerase-associated genes. Our data adds CVID to the broad clinical spectrum of cryptic adult-onset TBD. As the molecular diagnosis greatly impacts patient management and treatment strategies, we advise inclusion of all TBD-associated genes—despite their low prevalence—into the molecular screening of patients with antibody deficiencies.

Distribution of Chromosome-Arm Specific Telomere Length in Patients with Telomere Biology Disorders

Introduction: Telomere biology disorders (TBD) are multisystem diseases often associated with bone marrow failure (BMF) and pulmonary fibrosis. TBD are clinically diagnosed when mean telomere lengths (TL) in blood cells measured by clinical flowFISH assays fall &amp;lt;1 st percentile, in comparison to age-matched reference controls. This information has limitations, e.g. TL in blood cells may not represent TL in other organs (e.g. lung, liver) and moreover, individuals with high clinical suspicion of TBD can show borderline average TL which complicates their diagnosis. The likely reason for this is that cell senescence can be triggered by shortening of a subset, or even a single chromosome arm telomere, a situation that may be missed when measuring average TL. Previous approaches to measure individual chromosome-arm telomere length (CA-TL) are only available for a small number of chromosome arms or can only estimate the number of shortened telomeres without matching them to specific chromosome arms. Taking advantage of recent developments in long-read next generation sequencing (NGS) and the availability of the recently published telomere-to-telomere (T2T) reference human genome, our group developed Telogator, (Stephens Z. et al, 2022) a bioinformatic tool that estimates CA-TL for most chromosome arms using long-read NGS data. Here we use Telogator to describe for the first time the CA-TL pattern in TBD patients in comparison with a control non-TBD cohort. Methods: TBD patients were identified through the Pre-Myeloid and Bone Marrow Failure Specialty Clinic at Mayo Clinic. All patients underwent flowFISH and genetic testing. Age-matched non-TBD patients identified at the Center for Individualized Medicine (Mayo Clinic) were used as reference controls. Informed consent was obtained from all individuals with approval from the Mayo Clinic Institutional Review Board. DNA from peripheral blood mononuclear cells was sequenced using PacBio Sequel II following the manufacturer indications for HiFi sequencing. BAM files were aligned to the T2T reference and CA-TL was calculated using Telogator as described in Stephens Z. et al. 2022. Average TL, CA-TL, and percent of short telomeres (both defined as &amp;lt;5 kb and &amp;lt;3.2 kb) were calculated. Comparisons of average TL and percent of short telomeres were performed using t-test, while Wilcoxon rank-sum test was used to compare CA-TL. Results: Four TBD patients [median age = 58.5 (52-60), 50% female] and 6 non-TBD controls [Median age = 56 (50-63), 33% female] were included in the analysis. All TBD patients presented average TL below the 1 st centile in both lymphocytes and granulocytes, and TBD-related phenotype (100% with pulmonary fibrosis, 50% with BMF, 25% with liver fibrosis). Three had a pathogenic variant in a TBD related genes [2 in TERT (NM_198253.3: c.2030G&amp;gt;A, p.(Gly677Asp) and c.2515_2533del, p.(Thr839Alafs*29)) and 1 in NHP2 (NM_017838.3: c.459_460delAT; p.(X154Argext29))]. The average TL calculated by Telogator showed high correlation with flowFISH values (r 2 = 0.9883, p = 0.0059) and was lower in TBD patients than controls (p = 0.0012). Next, when comparing individual CA-TL between groups, only half of CA-TL was statistically different in the TBD group compared to controls (1p, 3q, 4p, 5p, 5q, 6q, 7p, 7q, 8q, 9p, 9q, 10q, 11p, 12q, 12q, 16p, 16q, 18q, 19p, 19q, 20p, 20q, 21q - Figure 1). The percentage of telomeres &amp;lt;5kb and &amp;lt;3 kb was significantly increased in TBD patients compared to controls (p= 0.014 and 0.0333, respectively). Discussion: Using a novel informatic tool, we replicate previous results obtained using classic approaches and were able to expand on these studies by measuring specific CA-TL in TBD patients. We identified statistically significant differences at the CA-TL level in these patients when compared to age-matched controls, while other CA-TL were not statistically different. Future studies and the inclusion of additional TBD patients will help define the clinical relevance of the differential CA-TL shortening and its prevalence among TBD patients. While limited by the small number of samples in the study, we show the feasibility of using this approach to study TL with unprecedented resolution and show that Telogator offers important advantages compared to classic methods to study telomere biology that can be used in other relevant fields like aging and/or cancer. Our results pave the way to advance the mechanistic understanding of TBD.

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.

Telomere Length Testing of Lymphocytes Alone By Flow-FISH Is a Highly Sensitive and Specific Test to Screen for a Telomere Biology Disorder in a Cohort of Children and Very Young Adults with Bone Marrow Failure

Background Uncovering an underlying telomere biology disorder (TBD) in patients with bone marrow failure (BMF) has crucial implications for treatment and outcomes, including hematopoietic cell transplantation and malignancy risk. Telomere length (TL) testing of peripheral blood leukocytes by clinical Flow-FISH has become an important test in diagnosing TBDs. In seminal studies, Alter et al. compared age-adjusted TL lengths (&amp;lt; or ≥1 st percentile) in lymphocytes (lymphs), granulocytes (grans), and four lymph subsets in individuals with a known TBD and their unaffected relatives. They found that very low (VL) TL (&amp;lt;1 st percentile) in lymphs had a sensitivity (Sens) of 97%, specificity (Spec) of 91%, and positive predictive value (PPV) of 85% for a TBD diagnosis. VL TL in 3 or 4 lymph subsets had slightly better test performance with a Sens of 98%, Spec of 94%, and PPV of 89%. We hypothesized that test performance characteristics of flow FISH would be different if applied to a ‘real-world’ cohort, which could inform the use of TL in lymphs alone versus in the four lymph subsets for different clinical indications. Methods We performed a search of the electronic medical record to identify all individuals who had Flow-FISH performed at Texas Children's Hospital from 2007 - 2022 (Figure 1). We excluded those who had the 2-panel test (lymphs and grans) or family TL cascade testing. We collected the indication for and results of Flow-FISH, demographic and clinical characteristics, and genetic testing information via chart review. A composite score (Comp Score) was created to define TBD (Comp Score of 1 or 2) vs. Not TBD (Comp Score &amp;lt;1). Performance of lymph TL alone and TL in 3 or 4 lymph subsets in predicting TBD was evaluated using Sens, Spec, PPV, NPV, area under ROC curve (AUC), and Cohen's Kappa statistic (k). Results Our final cohort included 433 individuals who received the 6-panel test (lymphs, grans, and four lymph subsets) with a median age of 9.7 years, range 0.06-22.6. Top indications for Flow-FISH were severe aplastic anemia (SAA) (n=106), mild aplastic anemia (AA) (i.e., not fulfilling criteria for MAA or SAA) (103), cytopenias without known or documented bone marrow hypoplasia or dysplasia (70), moderate aplastic anemia (MAA) (37), non-therapy-related myelodysplastic syndrome (26), and an incidental TBD gene variant identified through comprehensive sequencing (17). Twenty-two subjects (5.1%) had a TBD, 17 (3.9%) of which had a pathogenic/likely pathogenic variant (P/LPV) in a known TBD gene (Figure 1). Seven of those with SAA (6.6%) had VL TL in lymphs, 5 (4.7%) of which also had VL TL in 3 or 4 of the lymph subsets, and only 1 (0.9%) had a TBD based on Comp Score. Importantly, none had VL TL in 3 or 4 of the lymph subsets without VL TL in lymphs. Five of those with MAA (13.5%) had VL TL in lymphs, all of which had VL TL in 3 or 4 lymph subsets. Three had a TBD based on Comp Score, and each had a PV in a TBD gene; a TBD variant was not identified in two despite exome sequencing and chromosomal microarray testing. Eighteen of those with mild AA (17.5%) had VL TL in lymphs, 15 (14.6%) also had VL TL in 3 or 4 lymph subsets, 9 (8.7%) had a TBD based on Comp Score, and 7 (6.8%) had a P/LPV in a TBD gene. The Sens of TL in lymphs &amp;lt; vs. ≥ 1 st percentile and TL &amp;lt; 1 st percentile in 3 or 4 vs. &amp;lt; 3 lymph subsets against TBD based on Comp Score in those with AA regardless of severity was 100%, and the Spec ranged from 90.4% (lymph TL testing alone in mild AA) to 96.2% (lymph subsets testing in SAA) (Table 1). The PPV for both test approaches was low (≤20%) in SAA due to the low prevalence of TBD in this population. PPV was between 50-60% in the other AA groups. The AUC for detecting a TBD in the various AA groups was comparably high for both test approaches (0.95-0.98). The two tests had a substantial to almost perfect agreement (k=0.82, p&amp;lt;0.001). Conclusions This single institutional cohort demonstrates that the prevalence of TBD in young patients presenting with SAA is remarkably low, and the Sens and Spec of TL testing in lymphs alone are sufficiently high to detect the rare TBD patient and prompt additional testing (e.g., genetics) in few patients due to false positive testing. The prevalence of TBD is higher in those with MAA or mild AA, yet both test approaches detect all TBD patients. Spec level in mild AA is marginally higher with lymph subset TL testing but, at ~94%, underscores the need for other clinical parameters (e.g., genetics) to establish a TBD diagnosis in a small percentage of patients.

Prevalence and Spectrum of Autoimmune Diseases Among Patients with Telomere Biology Disorder

Introduction Telomeres are the protective ends of linear chromosomes that prevent DNA degradation with each replication. Premature attrition or dysfunction of telomeres due to an inherited defect in a telomere maintenance gene results in a spectrum of phenotypes collectively known as telomere biology disorders (TBD). To date, 18 genes have been implicated in TBD. Disease phenotypes are variable including bone marrow failure, immunodeficiency, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), idiopathic pulmonary fibrosis (IPF), liver cirrhosis, nail dystrophy, and premature graying of hair. Short telomeres have been observed in patients with autoimmune disorders (AID) and have been shown to increase the risk of AID. Furthermore, TBD have been associated with T cell dysfunction and exhaustion, mechanisms which may predispose to autoimmune phenotypes. Given the potential relationship between short telomeres and immune dysregulation, we hypothesize that the prevalence of AID may be increased in patients with a diagnosis of TBD. The aim of this study is to describe the prevalence and spectrum of AID in a cohort of patients with TBD. Methods Health record data were reviewed for 34 adult patients with confirmed TBD treated at the University of Wisconsin-Madison. A TBD diagnosis was established for patients who had a compatible clinical history plus either critically short lymphocyte telomere lengths (&amp;lt;1 st percentile for age as measured by FlowFISH) or one or more pathogenic/likely pathogenic variant(s) in a gene known to cause TBD. Demographics, rheumatologic history, serologies, and family history were collected. AID were included if diagnoses were made by a rheumatologist or other appropriate specialist with clinical features, physical exam findings, and/or serologies meeting diagnostic criteria per subspecialty guidelines. Results Eight of 34 patients (23.5%) with TBD had a concomitant diagnosis of a primary autoimmune condition. Specific AID included inflammatory bowel disease, Graves' disease, Hashimoto's thyroiditis, rheumatoid arthritis, scleroderma, and Sjogren's syndrome (Table 1). Two patients had autoimmune cytopenias, including steroid-responsive immune thrombocytopenic purpura and pure red cell aplasia (PRCA). Patient 8 was diagnosed with PRCA mixed with spur cell anemia prior to transplant, and bone marrow biopsy showed pure erythroid hypoplasia without evidence of malignancy. He responded to treatment with steroids and immunosuppression. Of the eight patients with a primary autoimmune condition, two had pathogenic/like pathogenic variants, two had rare variants of uncertain significance, and four had no detectable variants in a telomere associated gene. Twenty-three patients (67.6%) had a first- or second-degree relative with an autoimmune disorder. Conclusions An AID, as defined by strict diagnostic criteria per subspecialty guidelines, was present in 23.5% of patients with TBD defined by conservative diagnostic criteria. The spectrum of AID was diverse, including two patients with autoimmune hematologic disorders, and the majority had a family history of various AID. Thus, TBD may be associated with an increased prevalence of AID, though confirmation in a larger cohort of genetically defined TBD patients is needed. In the absence of a causative genetic lesion, diagnosis of a TBD based on critically short telomeres in those with multisystem autoimmune disorders with features that overlap TBD phenotypes can be challenging. Screening for AID as part of routine TBD comprehensive assessments as well as offering germline genetic testing for individuals with primary autoimmune disease with a compatible TBD phenotypes and studies to determine the mechanistic relationship between telomere length and autoimmunity are warranted.

P012 - Telomere biology as a prognostic factor in radical prostatectomy

P012 - Telomere biology as a prognostic factor in radical prostatectomy

Telomere Dynamics in Livestock.

Telomeres are repeated sequences of nucleotides at the end of chromosomes. They deteriorate across mitotic divisions of a cell. In Homo sapiens this process of lifetime reduction has been shown to correspond with aspects of organismal aging and exposure to stress or other insults. The early impetus to characterize telomere dynamics in livestock related to the concern that aged donor DNA would result in earlier cell senescence and overall aging in cloned animals. Telomere length investigations in dairy cows included breed effects, estimates of additive genetic control (heritability 0.12 to 0.46), and effects of external stressors on telomere degradation across animal life. Evaluation of telomeres with respect to aging has also been conducted in pigs and horses, and there are fewer reports of telomere biology in beef cattle, sheep, and goats. There were minimal associations of telomere length with animal productivity measures. Most, but not all, work in livestock has documented an inverse relationship between peripheral blood cell telomere length and age; that is, a longer telomere length was associated with younger age. Because livestock longevity affects productivity and profitability, the role of tissue-specific telomere attrition in aging may present alternative improvement strategies for genetic improvement while also providing translational biomedical knowledge.

Vascular Aging and Telomere Biology: The Role of Vitamin D3 Deficiency

Rationale. The impact of vitamin D3 deficiency on the risk and prognosis of numerous chronic diseases has been actively studied for years. Recent research has demonstrated that vitamin D is not merely involved in the control of calcium-phosphorus metabolism, but can also enhance insulin sensitivity, decrease the incidence of type 2 diabetes mellitus (T2DM), obesity and autoimmune destruction of pancreatic β-cells. The influence of vitamin D3 on some cardiometabolic risk factors and cardiovascular disease (CVD) was described. Thus studying the role of vitamin D3 in the development of arteries wall changes in T2DM and IR, and their relationship with biology telomere seems to be quite relevant.Aim. To study the relationship between vitamin D3 deficiency and vascular wall condition, telomere biology in patients with varying insulin sensitivity.Materials and methods. The cross-sectional study involved 305 patients (106 men and 199 women) aged 51.5 ± 13.3 y.o. All patients underwent laboratory and instrumental research methods, study of morphofunctional state vascular wall. Telomere length and telomerase activity were determined using polymerase chain reaction.Results. Totally, 18 patients out of 248 (7.2%) were found to have normal vitamin D3 level (more than 30 ng/ml). In 92.8% of those studied Vitamin D3 insufficiency or deficiency was determined. As increase in vitamin D3 deficiency, an increase in fasting glucose was noted, HbA1c and its elevated concentration, HOMA index, glucose disorders up to T2DM, higher vascular stiffness. Telomerase activity in group with vitamin D3 deficiency was significantly lower than in groups with vitamin D3 insufficiency and normal content. Multiple linear regression analysis revealed that they are independently associated with vitamin D3 in T2DM (B=1.43; st. OR. 0.106; p=0.0001), vascular stiffness (B=0.075; st. OR. 2.11; p=0.017), fasting glucose (B=0.169; st. OR 1.62; p=0.004), HbA1c level (B=0.062; st. OR. 7.4; p=0.001) and the presence of “short” telomeres (B=0.09; st. OR. 1.154; p=0.001). ROC analysis revealed relationships between BMI (0.634, p=0.001), duration of T2DM (0.651, p=0.022), high intima media thickness (0.614, p=0.004), vascular stiffness (0.605, p&amp;lt;0.001), HbA1c (0.588, p=0.022) and presence of vitamin D3 deficiency.Conclusion. In persons with varying insulin sensitivity — from insulin resistance up T2DM is advisable assess vitamin D3 levels for effective prevention of arterial wall changes in addition to traditional CVD risk factors. Availability Vitamin D3 deficiency requires active prevention metabolic disorders and vascular changes.

Telomere dysfunction promotes cholangiocyte senescence and biliary fibrosis in Primary Sclerosing Cholangitis.

Cellular senescence and biliary fibrosis are prototypical features of obliterative cholangiopathies, such as Primary Sclerosing Cholangitis (PSC). Telomere dysfunction can lead to senescence either through telomere erosion or damaged telomeres. Our goal was to investigate a mechanistic relationship between telomere damage and biliary fibrosis in PSC. Telomere attrition was observed in the bile ducts of PSC patients along with a reduction in telomerase reverse transcriptase (TERT) expression compared to normal livers. Similarly, liver tissue from mice models of biliary fibrosis showed telomere attrition with increased damage at telomeres measured as telomere-associated foci (TAF). Cellular models of senescence induction increased the TAFs in cholangiocytes. This coincided with decreased TERT expression and increased senescence, which was rescued by modulating TERT levels. Epigenetic analysis revealed increased acquisition of repressive histone methylation at the TERT promoter which correlated with decreased TERT transcription. Cholangiocyte-selective deletion of TERT in mice exacerbated fibrosis whereas androgen therapy towards telomerase rescued liver fibrosis and liver function in genetic mouse model of PSC. Our results demonstrate a mechanistic role for telomere dysfunction in cellular senescence and fibrosis that characterize PSC. This suggests that PSC may be, in part, a telomere biology disorder, and identifies TERT as a potential therapeutic target.

Telomouse—a mouse model with human-length telomeres generated by a single amino acid change in RTEL1

Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.

Mitochondria and telomeres: hand in glove.

Born as an endosymbiont, the bacteria engulfed by the proto-eukaryotic cell more than 1.45billion years ago progressively evolved as an important organelle with multiple interactions with the host cell. In particular, strong connections between mitochondria and the chromosome ends, the telomeres, led to propose a new theory of ageing in which dysfunctional telomeres and mitochondria are the main actors of a vicious circle reducing cell fitness and promoting cellular ageing. We review the evidences that oxidative stress and dysfunctional mitochondria damage telomeres and further discuss the interrelationship between telomere biology and mitochondria through the lens of telomerase which shuttles between the nucleus and mitochondria. Finally, we elaborate on the possible role of the mitochondrial genome on the inheritance of human telomere length through the expression of mitochondrial gene variants.

Telomeric repeat-containing RNA is dysregulated in acute myeloid leukemia

AbstractTERRA (telomeric repeat-containing RNA) is a class of long noncoding RNAs transcribed from subtelomeric and telomeric regions. TERRA binds to the subtelomeric and telomeric DNA–forming R-loops (DNA-RNA hybrids), which are involved in telomere maintenance and telomerase function, but the role of TERRA in human cells is not well characterized. Here, we comprehensively investigated for the first time TERRA expression in primary human hematopoietic cells from an exploratory cohort of patients with acute myeloid leukemia (AML), patients with acute lymphoblastic leukemia (ALL), patients with telomere biology disorder (TBD), and healthy subjects. TERRA expression was repressed in primary human hematopoietic cells, including healthy donors, patients with ALL, and patients with TBD, irrespective of their telomere length, except for AML. A second cohort comprising 88 patients with AML showed that TERRA was overexpressed in an AML subgroup also characterized by higher R-loop formation, low TERT and RNAseH2 expression, and a paucity of somatic splicing factor mutations. Telomere length did not correlate with TERRA expression levels. To assess the role of TERRA R-loops in AML, we induced R-loop depletion by increasing RNAseH1 expression in 2 AML cell lines. Decreased TERRA R-loops in AML cell lines resulted in increased chemosensitivity to cytarabine. Our findings indicate that TERRA is uniformly repressed in primary human hematopoietic cells but abnormally expressed in an AML subset with low telomerase.

Germline landscape of RPA1, RPA2 and RPA3 variants in pediatric malignancies: identification of RPA1 as a novel cancer predisposition candidate gene.

Replication Protein A (RPA) is single-strand DNA binding protein that plays a key role in the replication and repair of DNA. RPA is a heterotrimer made of 3 subunits - RPA1, RPA2, and RPA3. Germline pathogenic variants affecting RPA1 were recently described in patients with Telomere Biology Disorders (TBD), also known as dyskeratosis congenita or short telomere syndrome. Premature telomere shortening is a hallmark of TBD and results in bone marrow failure and predisposition to hematologic malignancies. Building on the finding that somatic mutations in RPA subunit genes occur in ~1% of cancers, we hypothesized that germline RPA alterations might be enriched in human cancers. Because germline RPA1 mutations are linked to early onset TBD with predisposition to myelodysplastic syndromes, we interrogated pediatric cancer cohorts to define the prevalence and spectrum of rare/novel and putative damaging germline RPA1, RPA2, and RPA3 variants. In this study of 5,993 children with cancer, 75 (1.25%) harbored heterozygous rare (non-cancer population allele frequency (AF) < 0.1%) variants in the RPA heterotrimer genes, of which 51 cases (0.85%) had ultra-rare (AF < 0.005%) or novel variants. Compared with Genome Aggregation Database (gnomAD) non-cancer controls, there was significant enrichment of ultra-rare and novel RPA1, but not RPA2 or RPA3, germline variants in our cohort (adjusted p-value < 0.05). Taken together, these findings suggest that germline putative damaging variants affecting RPA1 are found in excess in children with cancer, warranting further investigation into the functional role of these variants in oncogenesis.

Genetic Counseling and Family Screening Recommendations in Patients with Telomere Biology Disorders.

Telomere biology disorders (TBDs) encompass a spectrum of genetic diseases with a common pathogenesis of defects in telomerase function and telomere maintenance causing extremely short telomere lengths. Here, we review the current literature surrounding genetic testing strategies, cascade testing, reproductive implications, and the role of genetic counseling. The understanding of the genetic causes and clinical symptoms of TBDs continues to expand while genetic testing and telomere length testing are nuanced tools utilized in the diagnosis of this condition. Access to genetic counseling is becoming more abundant and is valuable in supporting patients and their families in making informed decisions. Patient resources and support groups are valuable to this community. Defining which populations should be offered genetic counseling and testing is imperative to provide proper diagnoses and medical management for not only the primary patient, but also their biological relatives.

Associations among temperament characteristics and telomere length and attrition rate in early childhood.

There is growing interest in telomere length as an indicator of current and future health. Although early childhood is a period of rapid telomere attrition, little is known about the factors that influence telomere biology during this time. Adult research suggests that telomere length is influenced by psychological characteristics. This study's goal was to test associations among repeated measures of temperament and telomere length in a community sample of children (N = 602; 52% male, 73% non-Hispanic White, middle-to-high socioeconomic status) from infancy to age 3 years. Relative telomere length was assessed from DNA in saliva samples collected at infancy (M = 8.4 months), 2 years (M = 24.9 months), and 3 years (M = 37.8 months). Temperament was assessed via maternal report questionnaires administered at infancy (Infant Behavior Report Questionnaire-Revised) and ages 2 and 3 years (Early Childhood Behavior Questionnaire). Temperament was operationalized in two ways: using the established domains of negative affectivity, surgency/extraversion, and regulation/effortful control and using person-centered scores that identified three groups of children with similar profiles across domains (emotionally and behaviorally regulated; emotionally and behaviorally dysregulated; introverted and overcontrolled). Analyses revealed that greater regulation/effortful control was associated with longer telomere length across time points. Additionally, higher surgency/extraversion, beginning in infancy, was associated with decreased rate of telomere attrition. There were no sex differences in the relations between temperament and telomere measures. These findings suggest that, as early as infancy, temperament may influence telomere biology, with a potential protective effect of positive temperament characteristics on telomere erosion. (PsycInfo Database Record (c) 2023 APA, all rights reserved).