Antibody signatures against viruses and microbiome reflect past and chronic exposures and associate with aging and inflammation.

Encounters with pathogens and other molecules can imprint long-lasting effects on our immune system, influencing future physiological outcomes. Given the wide range of microbes to which humans are exposed, their collective impact on health is not fully understood. To explore relations between exposures and biological aging and inflammation, we profiled an antibody-binding repertoire against 2,815 microbial, viral, and environmental peptides in a population cohort of 1,443 participants. Utilizing antibody-binding as a proxy for past exposures, we investigated their impact on biological aging, cell composition, and inflammation. Immune response against cytomegalovirus (CMV), rhinovirus, and gut bacteria relates with telomere length. Single-cell expression measurements identified an effect of CMV infection on the transcriptional landscape of subpopulations of CD8 and CD4 T-cells. This examination of the relationship between microbial exposures and biological aging and inflammation highlights a role for chronic infections (CMV and Epstein-Barr virus) and common pathogens (rhinoviruses and adenovirus C).

Genetic, parental and lifestyle factors influence telomere length.

The average length of telomere repeats (TL) declines with age and is considered to be a marker of biological ageing. Here, we measured TL in six blood cell types from 1046 individuals using the clinically validated Flow-FISH method. We identified remarkable cell-type-specific variations in TL. Host genetics, environmental, parental and intrinsic factors such as sex, parental age, and smoking are associated to variations in TL. By analysing the genome-wide methylation patterns, we identified that the association of maternal, but not paternal, age to TL is mediated by epigenetics. Single-cell RNA-sequencing data for 62 participants revealed differential gene expression in T-cells. Genes negatively associated with TL were enriched for pathways related to translation and nonsense-mediated decay. Altogether, this study addresses cell-type-specific differences in telomere biology and its relation to cell-type-specific gene expression and highlights how perinatal factors play a role in determining TL, on top of genetics and lifestyle.

Sequential Stem Cell-Kidney Transplantation in Schimke Immuno-osseous Dysplasia.

Lifelong immunosuppression is required for allograft survival after kidney transplantation but may not ultimately prevent allograft loss resulting from chronic rejection. We developed an approach that attempts to abrogate immune rejection and the need for post-transplantation immunosuppression in three patients with Schimke immuno-osseous dysplasia who had both T-cell immunodeficiency and renal failure. Each patient received sequential transplants of αß T-cell-depleted and CD19 B-cell-depleted haploidentical hematopoietic stem cells and a kidney from the same donor. Full donor hematopoietic chimerism and functional ex vivo T-cell tolerance was achieved, and the patients continued to have normal renal function without immunosuppression at 22 to 34 months after kidney transplantation. (Funded by the Kruzn for a Kure Foundation.).

Telomere length and epigenetic clocks as markers of cellular aging: a comparative study.

Telomere length (TL) and DNA methylation-based epigenetic clocks are markers of biological age, but the relationship between the two is not fully understood. Here, we used multivariable regression models to evaluate the relationships between leukocyte TL (LTL; measured by qPCR [n = 635] or flow FISH [n = 144]) and five epigenetic clocks (Hannum, DNAmAge pan-tissue, PhenoAge, SkinBlood, or GrimAge clocks), or their epigenetic age acceleration measures in healthy adults (age 19-61 years). LTL showed statistically significant negative correlations with all clocks (qPCR: r = - 0.26 to - 0.32; flow FISH: r = - 0.34 to - 0.49; p < 0.001 for all). Yet, models adjusted for age, sex, and race revealed significant associations between three of five clocks (PhenoAge, GrimAge, and Hannum clocks) and LTL by flow FISH (p < 0.01 for all) or qPCR (p < 0.001 for all). Significant associations between age acceleration measures for the same three clocks and qPCR or flow FISH TL were also found (p < 0.01 for all). Additionally, LTL (by qPCR or flow FISH) showed significant associations with extrinsic epigenetic age acceleration (EEAA: p < 0.0001 for both), but not intrinsic epigenetic age acceleration (IEAA; p > 0.05 for both). In conclusion, the relationships between LTL and epigenetic clocks were limited to clocks reflecting phenotypic age. The observed association between LTL and EEAA reflects the ability of both measures to detect immunosenescence. The observed modest correlations between LTL and epigenetic clocks highlight a possible benefit from incorporating both measures in understanding disease etiology and prognosis.

Short NK- and Naïve T-Cell Telomere Length Is Associated with Thyroid Cancer in Childhood Cancer Survivors: A Report from the Childhood Cancer Survivor Study.

BACKGROUND: Survivors of childhood cancer are at risk for therapy-related subsequent malignant neoplasms (SMN), including thyroid SMN. Telomere length (TL) is associated with cancer risk, but the relationship between TL and SMN risk among survivors is less clear. METHODS: We conducted a nested, matched case-control study of radiation-exposed 15-year+ adult survivors of childhood cancer with thyroid SMN (cases) and without SMN (controls). Forty-six cases were matched to 46 controls by primary diagnosis, chemotherapy (yes/no), radiation field, and follow-up duration. Lymphocyte TL (LTL) was measured by telomere flow-FISH cytometry using blood samples banked at a mean of 38.9 years (cases), 39.2 years (controls). Genetic variation in telomere genes was assessed by whole genome sequencing. Point estimates for LTL <10th percentile were determined for cases and controls. RESULTS: Cases had shorter median LTL than controls in three out of four leukocyte subsets. Cases were more likely to have NK cell LTL <10th percentile (P = 0.01), and 2.8-fold more likely to have naïve T-cell LTL <10th percentile than controls (CI, 1.07-8.78). Five out of 15 cases with a rare indel or missense variant had naïve T-cell LTL <10th percentile, compared with one out of eight controls. CONCLUSIONS: Long-term survivors have shorter than expected LTL, a finding that is more pronounced among survivors with thyroid SMN. IMPACT: The long-term impact of childhood cancer treatment on immune function is poorly understood. Our findings support immune function studies in larger survivor cohorts to assess long-term deficits in adaptive and innate immunity that may underlie SMN risk.

Fat metabolism is associated with telomere length in six population-based studies.

Telomeres are repetitive DNA sequences located at the end of chromosomes, which are associated to biological aging, cardiovascular disease, cancer and mortality. Lipid and fatty acid metabolism have been associated with telomere shortening. We have conducted an in-depth study investigating the association of metabolic biomarkers with telomere length (LTL). We performed an association analysis of 226 metabolic biomarkers with LTL using data from 11 775 individuals from six independent population-based cohorts (BBMRI-NL consortium). Metabolic biomarkers include lipoprotein lipids and subclasses, fatty acids, amino acids, glycolysis measures and ketone bodies. LTL was measured by quantitative polymerase chain reaction or FlowFISH. Linear regression analysis was performed adjusting for age, sex, lipid-lowering medication and cohort-specific covariates (model 1) and additionally for body mass index (BMI) and smoking (model 2), followed by inverse variance-weighted meta-analyses (significance threshold Pmeta = 6.5 × 10-4). We identified four metabolic biomarkers positively associated with LTL, including two cholesterol to lipid ratios in small VLDL (S-VLDL-C % and S-VLDL-CE %) and two omega-6 fatty acid ratios (FAw6/FA and LA/FA). After additionally adjusting for BMI and smoking, these metabolic biomarkers remained associated with LTL with similar effect estimates. In addition, cholesterol esters in very small VLDL (XS-VLDL-CE) became significantly associated with LTL (P = 3.6 × 10-4). We replicated the association of FAw6/FA with LTL in an independent dataset of 7845 individuals (P = 1.9 × 10-4). To conclude, we identified multiple metabolic biomarkers involved in lipid and fatty acid metabolism that may be involved in LTL biology. Longitudinal studies are needed to exclude reversed causation.

DNA-methylation-based telomere length estimator: comparisons with measurements from flow FISH and qPCR.

Telomere length (TL) is a marker of biological aging associated with several health outcomes. High throughput reproducible TL measurements are needed for large epidemiological studies. We compared the novel DNA methylation-based estimator (DNAmTL) with the high-throughput quantitative PCR (qPCR) and the highly accurate flow cytometry with fluorescent in situ hybridization (flow FISH) methods using blood samples from healthy adults. We used Pearson's correlation coefficient, Bland Altman plots and linear regression models for statistical analysis. Shorter DNAmTL was associated with older age, male sex, white race, and cytomegalovirus seropositivity (p<0.01 for all). DNAmTL was moderately correlated with qPCR TL (N=635, r=0.41, p < 0.0001) and flow FISH total lymphocyte TL (N=144, r=0.56, p < 0.0001). The agreements between flow FISH TL and DNAmTL or qPCR were acceptable but with wide limits of agreement. DNAmTL correctly classified >70% of TL categorized above or below the median, but the accuracy dropped with increasing TL categories. The ability of DNAmTL to detect associations with age and other TL-related factors in the absence of strong correlation with measured TL may indicate its capture of aspects of telomere maintenance mechanisms and not necessarily TL. The inaccuracy of DNAmTL prediction should be considered during data interpretation and across-study comparisons.

Establishing a Framework for the Clinical Translation of Germline Findings in Precision Oncology.

Inherited genetic variation has important implications for cancer screening, early diagnosis, and disease prognosis. A role for germline variation has also been described in shaping the molecular landscape, immune response, microenvironment, and treatment response of individual tumors. However, there is a lack of consensus on the handling and analysis of germline information that extends beyond known or suspected cancer susceptibility in large-scale cancer genomics initiatives. As part of the Personalized OncoGenomics program in British Columbia, we performed whole-genome and transcriptome sequencing in paired tumor and normal tissues from advanced cancer patients to characterize the molecular tumor landscape and identify putative targets for therapy. Overall, our experience supports a multidisciplinary and integrative approach to germline data management. This includes a need for broader definitions and standardized recommendations regarding primary and secondary germline findings in precision oncology. Here, we propose a framework for identifying, evaluating, and returning germline variants of potential clinical significance that may have indications for health management beyond cancer risk reduction or prevention in patients and their families.

Genetic variation in POT1 and risk of thyroid subsequent malignant neoplasm: A report from the Childhood Cancer Survivor Study.

BACKGROUND: Telomere length is associated with risk for thyroid subsequent malignant neoplasm in survivors of childhood cancer. Here, we investigated associations between thyroid subsequent malignant neoplasm and inherited variation in telomere maintenance genes. METHODS: We used RegulomeDB to annotate the functional impact of variants mapping to 14 telomere maintenance genes among 5,066 five-or-more year survivors who participate in the Childhood Cancer Survivor Study (CCSS) and who are longitudinally followed for incidence of subsequent cancers. Hazard ratios for thyroid subsequent malignant neoplasm were calculated for 60 putatively functional variants with minor allele frequency ≥1% in or near telomere maintenance genes. Functional impact was further assessed by measuring telomere length in leukocyte subsets. RESULTS: The minor allele at Protection of Telomeres-1 (POT1) rs58722976 was associated with increased risk for thyroid subsequent malignant neoplasm (adjusted HR = 6.1, 95% CI: 2.4, 15.5, P = 0.0001; Fisher's exact P = 0.001). This imputed SNP was present in three out of 110 survivors who developed thyroid cancer vs. 14 out of 4,956 survivors who did not develop thyroid cancer. In a subset of 83 survivors with leukocyte telomere length data available, this variant was associated with longer telomeres in B lymphocytes (P = 0.004). CONCLUSIONS: Using a functional variant approach, we identified and confirmed an association between a low frequency intronic regulatory POT1 variant and thyroid subsequent malignant neoplasm in survivors of childhood cancer. These results suggest that intronic variation in POT1 may affect key protein binding interactions that impact telomere maintenance and genomic integrity.

From incomplete penetrance with normal telomere length to severe disease and telomere shortening in a family with monoallelic and biallelic PARN pathogenic variants.

PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease.

Correction: TERT promoter mutation in adult granulosa cell tumor of the ovary.

The original version of this Article omitted the author Hannah van Meurs from the Department of Gynecology, Center for Gynecologic Oncology Amsterdam, Academic Medical Center, 1100 DD Amsterdam, The Netherlands. This has been corrected in both the PDF and HTML versions of the article.

Understanding the evolving phenotype of vascular complications in telomere biology disorders.

Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.

Telomere Length Calibration from qPCR Measurement: Limitations of Current Method.

Telomere length (TL) comparisons from different methods are challenging due to differences in laboratory techniques and data configuration. This study aimed to assess the validity of converting the quantitative polymerase chain reaction (qPCR) telomere/single copy gene (T/S) ratio to TL in kilobases (kb). We developed a linear regression equation to predict TL from qPCR T/S using flow cytometry with fluorescence in situ hybridization (flow FISH) TL data from 181 healthy donors (age range = 19⁻53) from the National Marrow Donor Program (NMDP) biorepository. TL measurements by qPCR and flow FISH were modestly correlated (R² = 0.56, p < 0.0001). In Bland-Altman analyses, individuals with the shortest (≤10th percentile) or longest (≥90th) flow FISH TL had an over- or under-estimated qPCR TL (bias = 0.89 and -0.77 kb, respectively). Comparisons of calculated TL from the NMDP samples and 1810 age- and sex-matched individuals from the National Health and Nutrition Examination Survey showed significant differences (median = 7.1 versus 5.8 kb, respectively, p < 0.0001). Differences in annual TL attrition were also noted (31 versus 13 bp/year, respectively, p = 0.02). Our results demonstrate that TL calculated in kb from qPCR T/S may yield biased estimates for individuals with the shortest or longest TL, those often of high clinical interest. We also showed that calculated TL in kb from qPCR data are not comparable across populations and therefore are not necessarily useful.

Donor telomere length and causes of death after unrelated hematopoietic cell transplantation in patients with marrow failure.

Previous studies have suggested that longer donor leukocyte telomere length (TL) is associated with improved survival after hematopoietic cell transplantation (HCT) in severe aplastic anemia (SAA). This study aimed to determine whether cell-specific lymphocyte TL is associated with certain post-HCT causes of death. We used flow cytometry and fluorescence in situ hybridization to measure TL in donor total lymphocytes and subsets: naïve enriched T cells (CD45RA+CD20-), memory enriched T cells (CD45RA-CD20-), natural killer (NK) fully differentiated T cells (CD45RA+CD57+), and B cells (CD45RA+CD20+). Competing risk survival regression was used for cause-specific death analyses. Clinical data and biospecimens were available from the Center for International Blood and Marrow Transplant Research database and biorepository. The study included 197 patients who underwent unrelated-donor HCT for SAA between 1988 and 2004. The median age at HCT was 15 years (range, 0.5-40 years), and the median follow-up was 5 years (range, <1 month to 20.7 years). Longer donor TL in all cell subsets was associated with lower risk of all-cause mortality (P < .01). In cause-specific mortality analyses, longer TL in B cells (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.46-0.87; P = .006) and possibly NK fully differentiated T cells (HR, 0.7; 95% CI, 0.51 to 0.97; P = .03) was associated with lower risk of infection-related death. Donor TL in other tested lymphocyte subsets was not statistically significantly associated with death resulting from graft-versus-host disease or graft failure (P > .05). However, a trend toward excess risk of graft-versus-host mortality was noted (HR for total lymphocyte TL, 1.26; P = .15). In conclusion, longer donor TL was associated with reduced rate of infection-related deaths after HCT for SAA.

TERT promoter mutation in adult granulosa cell tumor of the ovary.

The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, which is a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified as gain-of-function mutations that promote transcriptional activation of TERT in multiple cancers, such as melanoma and glioblastoma. A recent study investigating TERT promoter mutations in ovarian carcinomas found C228T and C250T mutations in 15.9% of clear cell carcinomas. However, it is unknown whether these mutations are frequent in other ovarian cancer subtypes, in particular, sex cord-stromal tumors including adult granulosa cell tumors. We performed whole-genome sequencing on ten adult granulosa cell tumors with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that adult granulosa cell tumors with mutated TERT promoter have increased expression of TERT mRNA and exhibited significantly longer telomeres compared to those with wild-type TERT promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary adult granulosa cell tumors (22%), 24 of 58 recurrent adult granulosa cell tumors (41%), and 1 of 22 other sex cord-stromal tumors (5%). There was a significant difference in overall survival between patients with TERT C228T promoter mutation in the primary tumors and those without it (p = 0.00253, log-rank test). In seven adult granulosa cell tumors, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggest that TERT C228T promoter mutations may have an important role in progression of adult granulosa cell tumors.

Correlation of Leukocyte Telomere Length Measurement Methods in Patients with Dyskeratosis Congenita and in Their Unaffected Relatives.

Several methods have been employed to measure telomere length (TL) in human studies. It has been difficult to directly compare the results from these studies because of differences in the laboratory techniques and output parameters. We compared TL measurements (TLMs) by the three most commonly used methods, quantitative polymerase chain reaction (qPCR), flow cytometry with fluorescence in situ hybridization (flow FISH) and Southern blot, in a cohort of patients with the telomere biology disorder dyskeratosis congenita (DC) and in their unaffected relatives (controls). We observed a strong correlation between the Southern blot average TL and the flow FISH total lymphocyte TL in both the DC patients and their unaffected relatives (R² of 0.68 and 0.73, respectively). The correlation between the qPCR average TL and that of the Southern blot method was modest (R² of 0.54 in DC patients and of 0.43 in unaffected relatives). Similar results were noted when comparing the qPCR average TL and the flow FISH total lymphocyte TL (R² of 0.49 in DC patients and of 0.42 in unaffected relatives). In conclusion, the strengths of the correlations between the three widely used TL assays (qPCR, flow FISH, and Southern blot) were significantly different. Careful consideration is warranted when selecting the method of TL measurement for research and for clinical studies.

Defects in lymphocyte telomere homeostasis contribute to cellular immune phenotype in patients with cartilage-hair hypoplasia.

BACKGROUND: Mutations in the long noncoding RNA RNase component of the mitochondrial RNA processing endoribonuclease (RMRP) give rise to the autosomal recessive condition cartilage-hair hypoplasia (CHH). The CHH disease phenotype has some overlap with dyskeratosis congenita, a well-known "telomere disorder." RMRP binds the telomerase reverse transcriptase (catalytic subunit) in some cell lines, raising the possibility that RMRP might play a role in telomere biology. OBJECTIVE: We sought to determine whether a telomere phenotype is present in immune cells from patients with CHH and explore mechanisms underlying these observations. METHODS: We assessed proliferative capacity and telomere length using flow-fluorescence in situ hybridization (in situ hybridization and flow cytometry) of primary lymphocytes from patients with CHH, carrier relatives, and control subjects. The role of telomerase holoenzyme components in gene expression and activity were assessed by using quantitative PCR and the telomere repeat amplification protocol from PBMCs and enriched lymphocyte cultures. RESULTS: Lymphocyte cultures from patients with CHH display growth defects in vitro, which is consistent with an immune deficiency cellular phenotype. Here we show that telomere length and telomerase activity are impaired in primary lymphocyte subsets from patients with CHH. Notably, telomerase activity is affected in a gene dose-dependent manner when comparing heterozygote RMRP carriers with patients with CHH. Telomerase deficiency in patients with CHH is not mediated by abnormal telomerase gene transcript levels relative to those of endogenous genes. CONCLUSION: These findings suggest that telomere deficiency is implicated in the CHH disease phenotype through an as yet unidentified mechanism.

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts.

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.