Clonal landscape and clinical outcomes of telomere biology disorders: somatic rescuing and cancer mutations.

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development.

Differential diagnosis of bone marrow failure syndromes guided by machine learning.

The choice to postpone treatment while awaiting genetic testing can result in significant delay in definitive therapies in patients with severe pancytopenia. Conversely, the misdiagnosis of inherited bone marrow failure (BMF) can expose patients to ineffectual and expensive therapies, toxic transplant conditioning regimens, and inappropriate use of an affected family member as a stem cell donor. To predict the likelihood of patients having acquired or inherited BMF, we developed a 2-step data-driven machine-learning model using 25 clinical and laboratory variables typically recorded at the initial clinical encounter. For model development, patients were labeled as having acquired or inherited BMF depending on their genomic data. Data sets were unbiasedly clustered, and an ensemble model was trained with cases from the largest cluster of a training cohort (n = 359) and validated with an independent cohort (n = 127). Cluster A, the largest group, was mostly immune or inherited aplastic anemia, whereas cluster B comprised underrepresented BMF phenotypes and was not included in the next step of data modeling because of a small sample size. The ensemble cluster A-specific model was accurate (89%) to predict BMF etiology, correctly predicting inherited and likely immune BMF in 79% and 92% of cases, respectively. Our model represents a practical guide for BMF diagnosis and highlights the importance of clinical and laboratory variables in the initial evaluation, particularly telomere length. Our tool can be potentially used by general hematologists and health care providers not specialized in BMF, and in under-resourced centers, to prioritize patients for genetic testing or for expeditious treatment.

RecQ helicase expression in patients with telomeropathies.

BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.

Effects of nandrolone decanoate on telomere length and clinical outcome in patients with telomeropathies: a prospective trial.

Androgens have been reported to elongate telomeres in retrospective and prospective trials with patients with telomeropathies, mainly with bone marrow failure. In our single-arm prospective clinical trial (clinicaltrials gov. Identifier: NCT02055456), 17 patients with short telomeres and/or germline pathogenic variants in telomere biology genes associated with at least one cytopenia and/or radiologic diagnosis of interstitial lung disease were treated with 5 mg/kg of intramuscular nandrolone decanoate every 15 days for 2 years. Ten of 13 evaluable patients (77%) showed telomere elongation at 12 months by flow-fluorescence in situ hybridization (average increase, 0.87 kb; 95% confidence interval: 0.20-1.55 kb; P=0.01). At 24 months, all ten evaluable patients showed telomere elongation (average increase, 0.49 kb; 95% confidence interval: 0.24-1.23 kb; P=0.18). Hematologic response was achieved in eight of 16 patients (50%) with marrow failure at 12 months, and in ten of 16 patients (63%) at 24 months. Seven patients had interstitial lung disease at baseline, and two and three had pulmonary response at 12 and 24 months, respectively. Two patients died due to pulmonary failure during treatment. In the remaining evaluable patients, the pulmonary function remained stable or improved, but showed consistent decline after cessation of treatment. Somatic mutations in myeloid neoplasm-related genes were present in a minority of patients and were mostly stable during drug treatment. The most common adverse events were elevations in liver function test levels in 88%, acne in 59%, and virilization in 59%. No adverse events grade ≥4 was observed. Our findings indicate that nandrolone decanoate elongates telomeres in patients with telomeropathies, which correlated with clinical improvement in some cases and tolerable adverse events.

Eltrombopag preferentially expands haematopoietic multipotent progenitors in human aplastic anaemia.

Eltrombopag has been added to first-line treatment of immune aplastic anaemia (AA), resulting in higher responses. We analysed marrow samples of AA patients who responded to immunosuppressive therapy (IST) alone or in combination with eltrombopag for the composition of the haematopoietic stem and progenitor cell (HSPC) compartment. The number of CD34+ cells and multipotent progenitors was higher in patients treated with eltrombopag (P < 0·005; P < 0·05; respectively), but not the number of stem cells. No aberrant phenotype was observed. These results indicate that eltrombopag augments CD34+ cells in vivo and preferentially expands multipotent progenitors, but not stem cells.

Pathogenic TERT promoter variants in telomere diseases.

PURPOSE: The 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. METHODS: We 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. RESULTS: Pathogenic -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. CONCLUSION: We 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.

Obesity contributes to telomere shortening in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial disorder and obesity occurs in 38% to 88% of these women. Although hyperandrogenism may contribute to telomere lengthening, increased body mass index (BMI) is associated with telomere erosion. We sought to compare leukocyte telomere length (LTL) in PCOS women with normal, overweight, and obese BMI. We evaluated the relationship between LTL and clinical variables of PCOS and inflammatory biomarkers independent of BMI. A total of 348 women (243 PCOS and 105 non-PCOS) were evaluated for anthropometric measures, total testosterone, androstenedione, estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), sex hormone-binding globulin (SHBG), free androgen index (FAI), fasting insulin and glycemia, lipid profile, homocysteine, C-reactive protein (CRP) and homeostatic model of insulin resistance (HOMA-IR). LTL was measured by qPCR. The PCOS group presented higher weight, waist circumference, BMI, testosterone, LH, fasting insulin, FAI, and HOMA-IR, and lower E2, SHBG, and fasting glycemia measures compared with the non-PCOS. When stratified by BMI, LTL was increased in all subgroups in PCOS compared to non-PCOS. However, in the PCOS group, LTL was lower in overweight (P = 0.0187) and obese (P = 0.0018) compared to normal-weight women. The generalized linear model showed that BMI, androstenedione, homocysteine, and CRP were associated with telomere biology. Women with PCOS had longer LTL, however, overweight or obesity progressively contributes to telomere shortening and may affect reproductive outcomes of PCOS, while androstenedione may increase LTL.

Telomeric repeat-containing RNA is dysregulated in acute myeloid leukemia.

TERRA (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.

Defective hematopoietic differentiation of immune aplastic anemia patient-derived iPSCs.

In acquired immune aplastic anemia (AA), pathogenic cytotoxic Th1 cells are activated and expanded, driving an immune response against the hematopoietic stem and progenitor cells (HSPCs) that provokes cell depletion and causes bone marrow failure. However, additional HSPC defects may contribute to hematopoietic failure, reflecting on disease outcomes and response to immunosuppression. Here we derived induced pluripotent stem cells (iPSCs) from peripheral blood (PB) erythroblasts obtained from patients diagnosed with immune AA using non-integrating plasmids to model the disease. Erythroblasts were harvested after hematologic response to immunosuppression was achieved. Patients were screened for germline pathogenic variants in bone marrow failure-related genes and no variant was identified. Reprogramming was equally successful for erythroblasts collected from the three immune AA patients and the three healthy subjects. However, the hematopoietic differentiation potential of AA-iPSCs was significantly reduced both quantitatively and qualitatively as compared to healthy-iPSCs, reliably recapitulating disease: differentiation appeared to be more severely affected in cells from the two patients with partial response as compared to the one patient with complete response. Telomere elongation and the telomerase machinery were preserved during reprogramming and differentiation in all AA-iPSCs. Our results indicate that iPSCs are a reliable platform to model immune AA and recapitulate clinical phenotypes. We propose that the immune attack may cause specific epigenetic changes in the HSPCs that limit adequate proliferation and differentiation.

Immunosenescence in chronic HIV infected patients impairs essential functions of their natural killer cells.

The HIV/AIDS pandemic still represents an important global health issue. There is no sterilizing cure, therefore a continuous treatment is necessary, which caused the emerged idea of HIV as a chronic inflammatory disease that may also affect healthy aging. Considering that the activation profile of some innate cells such as natural killer cells has previously been associated to HIV progression, it remains to be better defined this activation status of NK cells considering the time of HIV infection. In this study, we characterized NK cell phenotype and function during acute and chronic HIV infection and also investigated markers of immunosenescence in these cells. Our results showed that chronic infected patients remained with elevated levels of some plasma inflammatory molecules (IP-10, sCD14) and a concurrent expansion of the non-functional NK cell subset (CD3-CD56-CD16+). NK cells from the chronic infected group displayed an activated profile with higher levels of cytokines and chemokines production (TNF-α, IL-12, IFN-α2, IFN-γ, IL-6, RANTES, MCP-1, IL-10, IL-4 and IL-5). The production of these molecules was positively correlated to the time of infection. Moreover, we noted a possible association of higher global DNA methylation frequency of NK cells in two HIV patients in the advanced stage of disease. Chronic infected patients also showed a trend towards higher production of reactive oxygen species by their NK cells which altogether suggest the evolution of these cells to a senescent state that might be further evaluated.

Telomere Length and Telomerase Activity in Immature Oocytes and Cumulus Cells of Women with Polycystic Ovary Syndrome.

Metaphase II oocytes (MII) from polycystic ovary syndrome (PCOS) frequently have impaired oocyte competence. Since telomere maintenance is important for folliculogenesis, oocyte maturation, and early embryonic development, we sought to verify the implications of PCOS on telomere length and telomerase activity in immature oocytes and cumulus cells. 43 PCOS and 67 control women were included, and anthropometric, biochemical, and hormonal characteristics were evaluated. The telomere length in germinal vesicle stage (GV) and in metaphase I (MI) oocytes, as well as in the cumulus cells of immature (CCI) and mature oocytes (CCM), and in leukocytes was measured by qPCR. The telomerase activity in reproductive cells was evaluated by the TRAPeze® XL Kit. The body mass index (p = 0.001), LH (p = 0.015), estradiol (p = 0.004), insulin (p = 0.002), testosterone (p < 0.0001), androstenedione (p = 0.001), free androgen index (p < 0.0001), and c-reactive protein (p = 0.003) were greater, while the FSH (p = 0.0002) was lower in the PCOS group. The telomere length in the CCI (p = 0.649) and CCM (p = 0.378) did not differ between the PCOS and the control groups. On the other hand, telomerase activity in the CCI (p = 0.003) and CCM (p = 0.022) was higher in the PCOS group. In the leukocyte's cells, the telomere length was reduced in the PCOS group (p = 0.025). In the GV and MI oocytes, no differences were observed in telomere length and telomerase activity between the groups. We showed that telomere length is not altered in reproductive cells from PCOS. However, higher telomerase activity in the CCI and CCM may be required for telomere length maintenance.

Telomere dynamics and hematopoietic differentiation of human DKC1-mutant induced pluripotent stem cells.

Telomeropathies are a group of phenotypically heterogeneous diseases molecularly unified by pathogenic mutations in telomere-maintenance genes causing critically short telomeres. X-linked dyskeratosis congenita (DC), the prototypical telomere disease, manifested with ectodermal dysplasia, cancer predisposition, and severe bone marrow failure, is caused by mutations in DKC1, encoding a protein responsible for telomerase holoenzyme complex stability. To investigate the effects of pathogenic DKC1 mutations on telomere repair and hematopoietic development, we derived induced pluripotent stem cells (iPSCs) from fibroblasts of a DC patient carrying the most frequent mutation: DKC1 p.A353V. Telomeres eroded immediately after reprogramming in DKC1-mutant iPSCs but stabilized in later passages. The telomerase activity of mutant iPSCs was comparable to that observed in human embryonic stem cells, and no evidence of alternative lengthening of telomere pathways was detected. Hematopoietic differentiation was carried out in DKC1-mutant iPSC clones that resulted in increased capacity to generate hematopoietic colony-forming units compared to controls. Our study indicates that telomerase-dependent telomere maintenance is defective in pluripotent stem cells harboring DKC1 mutation and unable to elongate telomeres, but sufficient to maintain cell proliferation and self-renewal, as well as to support the primitive hematopoiesis, the program that is recapitulated with our differentiation protocol.

Skewed X-chromosome inactivation and shorter telomeres associate with idiopathic premature ovarian insufficiency.

OBJECTIVE: To analyze whether telomere length, X-chromosome inactivation (XCI), and androgen receptor (AR) GAG polymorphism are related to idiopathic premature ovarian insufficiency (POI). DESIGN: Case-control study. SETTING: University hospital. PATIENT(S): A total of 121 women, including 46 nonsyndromic POI and 75 controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Age, weight, height, body mass index (BMI), systolic and diastolic arterial pressure, E2, androstenedione, T, and C-reactive protein were assessed. Telomere length was estimated by quantitative real-time polymerase chain reaction, XCI was measured using the Human Androgen Receptor and X-linked retinitis pigmentosa 2 (RP2) methylation assays. AR and FMR1 polymorphism was assessed by quantitative fluorescent polymerase chain reaction and sequencing. RESULT(S): Premature ovarian insufficiency women had a higher mean age, weighed less, and exhibited lower C-reactive protein, E2, and androstenedione levels. The AR polymorphism did not differ between the groups. Four patients had premutation (55-200 CGG repeats), and none displayed a full mutation in the FMR1 gene. However, patients with POI showed shorter telomere length and higher frequency of skewed XCI. Extreme skewing (≥90%) was observed in 15% of women with POI, and shorter telomeres correlated with XCI skewing in both groups. CONCLUSION(S): Skewed XCI and shortened telomere length were associated with idiopathic POI, despite no alterations in the AR and FMR1 genes. Additionally, there is a tendency for women with short telomeres to exhibit skewed XCI.

Telomere length correlates with disease severity and inflammation in sickle cell disease.

BACKGROUND: Telomeres, the ends of linear chromosomes, shorten during mitotic cell division and erosion may be aggravated by inflammation or proliferative and oxidative stress. As the bone marrow is under hyperproliferative pressure in sickle cell disease and several tissues are submitted to chronic inflammation, this study sought to determine the telomere length of patients with sickle cell disease. METHODS: The mean telomere length was measured in peripheral blood leukocytes by quantitative polymerase chain reaction. The age-adjusted telomere to single copy gene ratio was compared between 91 adult sickle cell disease patients and 188 controls. RESULTS: Sickle cell disease patients had significantly shorter telomeres than the controls (p-value<0.0001). Moreover, among sickle cell disease genotypes, Hb SS patients had significantly shorter telomeres compared to Hb SC and Hb Sß patients (p-value<0.0001). Patients on hydroxyurea also had shorter telomeres in comparison to those off the drug (p-value=0.02). A positive correlation was observed between telomere length and hemoglobin level (r=0.3; p-value=0.004), whereas negative correlations were detected between telomere length and lymphocyte count (r=-0.3; p-value=0.005) and interleukin-8 serum levels (r=-0.4; p-value=0.02). CONCLUSIONS: The findings of this study indicate that telomeres are short in sickle cell disease patients and that telomere erosion directly correlates with disease genotype, inflammation markers, and the use of hydroxyurea.

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma.

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

Telomere length correlates with disease severity and inflammation in sickle cell disease

ABSTRACT Background: Telomeres, the ends of linear chromosomes, shorten during mitotic cell division and erosion may be aggravated by inflammation or proliferative and oxidative stress. As the bone marrow is under hyperproliferative pressure in sickle cell disease and several tissues are submitted to chronic inflammation, this study sought to determine the telomere length of patients with sickle cell disease. Methods: The mean telomere length was measured in peripheral blood leukocytes by quantitative polymerase chain reaction. The age-adjusted telomere to single copy gene ratio was compared between 91 adult sickle cell disease patients and 188 controls. Results: Sickle cell disease patients had significantly shorter telomeres than the controls (p-value < 0.0001). Moreover, among sickle cell disease genotypes, Hb SS patients had significantly shorter telomeres compared to Hb SC and Hb Sβ patients (p-value < 0.0001). Patients on hydroxyurea also had shorter telomeres in comparison to those off the drug (p-value = 0.02). A positive correlation was observed between telomere length and hemoglobin level (r = 0.3; p-value = 0.004), whereas negative correlations were detected between telomere length and lymphocyte count (r = -0.3; p-value = 0.005) and interleukin-8 serum levels (r = -0.4; p-value = 0.02). Conclusions: The findings of this study indicate that telomeres are short in sickle cell disease patients and that telomere erosion directly correlates with disease genotype, inflammation markers, and the use of hydroxyurea.