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.

Disease progression and clinical outcomes in telomere biology disorders.

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

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.

Epigenetic Aging and Hematopoietic Cell Transplantation in Patients With Severe Aplastic Anemia.

Cellular aging in hematopoietic cell transplantation (HCT) is important in the context of immune reconstitution and age-related complications. Recently, several DNA-methylation (DNAm)-based biomarkers of aging known as "epigenetic clocks" have been introduced as novel tools to predict cellular age. Here, we used Cox proportional hazards models to assess the possible associations of donor pre-HCT DNAm age, and its post-HCT changes, using the recently published lifespan-associated epigenetic clock known as "DNAm-GrimAge," with outcomes among patients with severe aplastic anemia (SAA). The study included 732 SAA patients from the Transplant Outcomes in Aplastic Anemia project, who underwent unrelated donor HCT and for whom a donor pre-HCT blood DNA sample was available; 41 also had a post-HCT sample collected at day 100. In multivariable analyses, we found similar associations for donor chronological age and pre-HCT DNAm-GrimAge with post-HCT survival (hazard ratio [HR] per decade = 1.13; 95% confidence interval [CI], 0.99-1.28; P = .07 and HR = 1.14; 95% CI, 0.99-1.28; P = .06, respectively). In donors with 10+ years of GrimAge acceleration (ie, deviation from expected DNAm age for chronological age), elevated risks of chronic graft versus host disease (HR = 2.4; 95% CI, 1.21-4.65; P = .01) and possibly post-HCT mortality (HR = 1.79; 95% CI, 0.96-3.33; P = .07) were observed. In the subset with post-HCT samples, we observed a significant increase in DNAm-GrimAge in the first 100 days after HCT (median change 12.5 years, range 1.4 to 26.4). Higher DNAm-GrimAge after HCT was associated with inferior survival (HR per year = 1.11; 95% CI, 1.02-1.21; P = .01), predominantly within the first year after HCT. This study highlights the possible role cellular aging may play in HCT outcomes.

Leukocyte telomere length in patients with myotonic dystrophy type I: a pilot study.

Myotonic dystrophy type I (DM1) is an autosomal dominant disease of which clinical manifestations resemble premature aging. We evaluated the contribution of telomere length in pathogenesis in 361 DM1 patients (12 with serial measurements) and 223 unaffected relative controls using qPCR assay. While no differences in baseline leukocyte relative telomere length (RTL) was noted, the data suggested an accelerated RTL attrition in DM1 (discovery cohort: T/S change/year = -0.013 in DM1 vs. -0.005 in controls, P = 0.04); similar trend was noted in validation cohort. Further investigations are needed to examine the role of TL in the pathophysiology of DM1.

Diabetes, metformin and cancer risk in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is an autosomal dominant multisystem disorder characterized by myotonia and muscle weakness. Type 2 diabetes (T2D) and cancer have been shown to be part of the DM1 phenotype. Metformin, a well-established agent for the management of T2D, is thought to have cancer-preventive effects in the general population. In our study, we aimed to assess the association between T2D, metformin use and the risk of cancer in DM1 patients. We identified a cohort of 913 DM1 patients and an age-, sex- and clinic-matched cohort of 12,318 DM1-free controls from the UK Clinical Practice Research Datalink, a large primary care records database. We used Cox regression models to assess cancer risk in T2D patients who were metformin users or nonusers compared to patients without T2D. Separate analyses were conducted for DM1 patients and controls. T2D was more prevalent in DM1 than in controls (8% vs. 3%, p < 0.0001). DM1 patients with T2D, compared to those without T2D, were more likely to develop cancer (hazard ratio [HR] = 3.60, 95% confidence interval [CI] = 1.18-10.97; p = 0.02), but not if they were treated with metformin (HR = 0.43, 95% CI = 0.06-3.35; p = 0.42). Among controls, we observed no significant associations between T2D and cancer risk in either users or nonusers of Metformin (HR = 1.28, 95% CI = 0.91-1.79; p = 0.16 and HR = 1.13, 95% CI = 0.72-1.79; p = 0.59, respectively). These results show an association between T2D and cancer risk in DM1 patients and may provide new insights into the possible benefits of Metformin use in DM1.

Reproductive Cancer Risk Factors in Women With Myotonic Dystrophy (DM): Survey Data From the US and UK DM Registries.

Introduction: Recent evidence demonstrates that women with myotonic dystrophy type 1 are at increased risk of reproductive organ tumors. However, studies of reproductive cancer risk factors in those patients are lacking. Methods: Using questionnaires, we collected and analyzed personal history information related to cancer risk factors from women enrolled in a UK and US registry for myotonic dystrophy (dystrophia myotonica; DM) patients. Results: The survey was completed by 242 DM type 1 (DM1) and 44 DM type 2 (DM2) women enrolled in the UK Registry (N = 124) and the US National Registry (N = 162). The mean age at DM1 diagnosis was 33.8 years (standard deviation, SD = 13.2) and for DM2 was 49.2 (SD = 13.0). Mean age at survey was 48.7 (SD = 12.8) and 59.1 years (SD = 12.8) for DM1 and DM2, respectively. There were no statistically significant differences between DM1 and DM2 regarding menstrual history or fertility-related factors. Yet, women with DM2 were more likely to have used menopausal hormone therapy (HT) than women with DM1 (52.3 vs. 22.1%, p < 0.0001), and more women with DM2 had a hysterectomy (53.5 vs. 29.5%, p < 0.01). These differences were not statistically significant after age adjustment (OR = 2.00, p = 0.08, and OR = 1.40, p = 0.38, respectively). The frequency of self-reported reproductive organ tumors was not significantly different comparing DM1 to DM2 (p = 0.28). However, the data suggested that women with DM2 appear to have a lower risk of malignant tumors compared to those with DM1 (OR = 0.72, p = 0.69). Discussion: Our study is the first to characterize a wide range of reproductive risk factors in women with DM. We observed no significant differences between DM1 and DM2 in the factors that were evaluated, which suggests that the known excesses of ovarian and endometrial cancer previously reported in women with DM1 cannot be attributed to greater prevalence of standard cancer-related reproductive risk factors. Larger studies evaluating the possible link between reproductive cancer risk factors and risk of tumors in women with DM are needed.

Benign tumors in myotonic dystrophy type I target disease-related cancer sites.

OBJECTIVES: Recent evidence showed that myotonic dystrophy type I (DM1) patients are at increased risk of certain cancers, but the risk of benign tumors is unknown. We compared the risk of benign tumors in DM1 patients with matched DM1-free individuals and assessed the association between benign tumors and subsequent cancers. METHODS: We identified 927 DM1 patients and 13,085 DM1-free individuals matched on gender, birth-year, clinic, and clinic-registration year from the UK Clinical Practice Research Datalink, a primary care records database. We used Cox regression models for statistical analyses. RESULTS: DM1 patients had elevated risks of thyroid nodules (Hazard Ratio [HR] = 10.4; 95% Confidence Interval [CI] = 3.91-27.52; P < 0.001), benign tumors of the brain or nervous system (HR = 8.4; 95% CI = 2.48-28.47; P < 0.001), colorectal polyps (HR = 4.3; 95% CI = 1.76-10.41; P = 0.001), and possibly uterine fibroids (HR = 2.7; 95% CI = 1.22-5.88; P = 0.01). Pilomatricomas and salivary gland adenomas occurred almost exclusively in DM1 patients (Fisher's exact P < 0.001). The HR for colorectal polyps was elevated in DM1 males but not in females (HR = 8.2 vs. 1.3, respectively; P-heterogeneity < 0.001), whereas endocrine and brain tumors occurred exclusively in females. The data suggested an association between benign tumors and subsequent cancer in classic DM1 patients (HR = 2.7; 95% CI = 0.93-7.59; P = 0.07). INTERPRETATION: Our study showed a similar site-specific benign tumor profile to that previously reported for DM1-associated cancers. The possible association between benign tumors and subsequent cancer in classic DM1 patients warrants further investigation as it may guide identifying patients at elevated risk of cancer. Our findings underscore the importance of following population-based screening recommendations in DM1 patients, for example, for colorectal cancer.

Statin use and reduced risk of biliary tract cancers in the UK Clinical Practice Research Datalink.

OBJECTIVE: To evaluate the association between statin use and risk of biliary tract cancers (BTC). DESIGN: This is a nested case-control study conducted in the UK Clinical Practice Research Datalink. We included cases diagnosed with incident primary BTCs, including cancers of the gall bladder, bile duct (ie, both intrahepatic and extrahepatic cholangiocarcinoma), ampulla of Vater and mixed type, between 1990 and 2017. For each case, we selected five controls who did not develop BTCs at the time of case diagnosis, matched by sex, year of birth, calendar time and years of enrolment in the general practice using incidence density sampling. Exposures were defined as two or more prescription records of statins 1 year prior to BTC diagnosis or control selection. ORs and 95% CIs for associations between statins and BTC overall and by subtypes were estimated using conditional logistic regression, adjusted for relevant confounders. RESULTS: We included 3118 BTC cases and 15 519 cancer-free controls. Current statin use versus non-use was associated with a reduced risk of all BTCs combined (adjusted OR=0.88, 95% CI 0.79 to 0.98). The reduced risks were most pronounced among long-term users, as indicated by increasing number of prescriptions (ptrend=0.016) and cumulative dose of statins (ptrend=0.008). The magnitude of association was similar for statin use and risk of individual types of BTCs. The reduced risk of BTCs associated with a record of current statin use versus non-use was more pronounced among persons with diabetes (adjusted OR=0.72, 95% CI 0.57 to 0.91). Among non-diabetics, the adjusted OR for current statin use versus non-use was 0.91 (95% CI 0.81 to 1.03, pheterogeneity=0.007). CONCLUSION: Compared with non-use of statins, current statin use is associated with 12% lower risk of BTCs; no association found with former statin use. If replicated, particularly in countries with a high incidence of BTCs, our findings could pave the way for evaluating the value of statins for BTC chemoprevention.

Cancer Risk in Myotonic Dystrophy Type I: Evidence of a Role for Disease Severity.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is an inherited trinucleotide repeat disorder in which specific cancers have been implicated as part of the disease phenotype. This study aimed to assess whether cancer risk in DM1 patients is modified by disease severity. METHODS: Using the United Kingdom Clinical Practice Research Datalink (primary care electronic medical records), we identified a cohort of 927 DM1 and a matched cohort of 13 085 DM1-free individuals between January 1, 1988 and February 29, 2016. We used Cox regression models to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) of organ-specific cancer risks. Analyses were stratified by age at DM1 diagnosis as a surrogate for disease severity. Statistical tests were two-sided. RESULTS: Patients with classic DM1 (age at diagnosis: 11-40 years) were at elevated risk of cancer overall (HR = 1.81; 95% CI = 1.12 to 2.93); cancers of the thyroid (HR = 15.93; 95% CI = 2.45 to 103.64), uterus (HR = 26.76; 95% CI = 2.32 to 309.26), and cutaneous melanoma (HR = 5.98; 95% CI = 1.24 to 28.79) accounted for the excess. In late-onset DM1 patients (age at diagnosis >40 years), a reduced overall cancer risk was observed (HR = 0.53; 95% CI = 0.32 to 0.85), possibly driven by the deficit in hematological malignancies (DM1 = 0 cases, DM1-free = 54 cases; P = .02). The difference between the observed HR for classic and late-onset DM1 was statistically significant (P < .001). CONCLUSIONS: The observed difference in relative cancer risk between classic and late-onset DM1 patients compared with their DM1-free counterparts provides the first evidence that disease severity modifies DM1-related cancer susceptibility. This novel finding may guide clinical management and scientific investigations for the underlying molecular mechanisms in DM-related carcinogenesis.

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.

Quasi-experimental Studies in the Fields of Infection Control and Antibiotic Resistance, Ten Years Later: A Systematic Review.

OBJECTIVE A systematic review of quasi-experimental studies in the field of infectious diseases was published in 2005. The aim of this study was to assess improvements in the design and reporting of quasi-experiments 10 years after the initial review. We also aimed to report the statistical methods used to analyze quasi-experimental data. DESIGN Systematic review of articles published from January 1, 2013, to December 31, 2014, in 4 major infectious disease journals. METHODS Quasi-experimental studies focused on infection control and antibiotic resistance were identified and classified based on 4 criteria: (1) type of quasi-experimental design used, (2) justification of the use of the design, (3) use of correct nomenclature to describe the design, and (4) statistical methods used. RESULTS Of 2,600 articles, 173 (7%) featured a quasi-experimental design, compared to 73 of 2,320 articles (3%) in the previous review (P<.01). Moreover, 21 articles (12%) utilized a study design with a control group; 6 (3.5%) justified the use of a quasi-experimental design; and 68 (39%) identified their design using the correct nomenclature. In addition, 2-group statistical tests were used in 75 studies (43%); 58 studies (34%) used standard regression analysis; 18 (10%) used segmented regression analysis; 7 (4%) used standard time-series analysis; 5 (3%) used segmented time-series analysis; and 10 (6%) did not utilize statistical methods for comparisons. CONCLUSIONS While some progress occurred over the decade, it is crucial to continue improving the design and reporting of quasi-experimental studies in the fields of infection control and antibiotic resistance to better evaluate the effectiveness of important interventions. Infect Control Hosp Epidemiol 2018;39:170-176.

Benign and malignant tumors in the UK myotonic dystrophy patient registry.

INTRODUCTION: In light of recent evidence indicating that cancer is part of the myotonic dystrophy (DM) phenotype, we assessed the prevalence of benign and malignant tumors among 220 patients enrolled in the UK Myotonic Dystrophy Patient Registry and evaluated factors associated with their development. METHODS: A survey was distributed to collect tumor history and lifestyle information. We used multinomial logistic regression for the analysis. RESULTS: Thirty-nine benign (30 patients), and 16 malignant (15 patients) tumors were reported. Increasing age (odds ratio [OR] = 1.13, 95% confidence interval [CI] = 1.05-1.21, P = 0.001) and earlier age at DM diagnosis (OR = 1.06, 95% CI = 1.00-1.13, P = 0.04) were associated with benign and malignant tumors (OR = 1.20, 95% CI = 1.10-1.30, P < 0.001 and OR = 1.08, 95% CI = 1.01-1.15, P = 0.02, respectively). Female gender was associated with benign tumors only (OR = 6.43, 95% CI = 1.79-23.04, P = 0.004). No associations were observed between tumors and smoking (P = 0.24), alcohol consumption (P = 0.50), or body mass index (P = 0.21). DISCUSSION: Our results confirm previous findings suggesting a limited role for common lifestyle factors and a potential genetic contribution in DM tumor predisposition. Muscle Nerve 57: 316-320, 2018.

Risk of skin cancer among patients with myotonic dystrophy type 1 based on primary care physician data from the U.K. Clinical Practice Research Datalink.

Myotonic dystrophy type 1 (DM1) is an inherited multisystem neuromuscular disorder caused by a CTG trinucleotide repeat expansion in the DMPK gene. Recent evidence documents that DM1 patients have an increased risk of certain cancers, but whether skin cancer risks are elevated is unclear. Using the U.K. Clinical Practice Research Datalink (CPRD), we identified 1,061 DM1 patients and 15,119 DM1-free individuals matched on gender, birth year (±2 years), attending practice and registration year (±1 year). We calculated the hazard ratios (HRs) and 95% confidence intervals (CIs) for the association of DM1 diagnosis with skin cancer risk using Cox proportional hazards models, for all skin cancers combined and by histological subtype. Follow-up started at the latest of the age at practice registration, DM1 diagnosis/control selection or January 1st 1988, and ended at the earliest of the age at first skin cancer diagnosis, death, transfer out of the practice, last date of data collection or the end of the CPRD record (October 31, 2016). During a median follow-up of 3.6 years, 35 DM1 patients and 108 matched DM1-free individuals developed a skin cancer. DM1 patients had an increased risk of skin cancer overall (HR = 5.44, 95% CI = 3.33-8.89, p < 0.0001), and basal cell carcinoma (BCC) (HR = 5.78, 95% CI = 3.36-9.92, p < 0.0001). Risks did not differ by gender, or age at DM1 diagnosis (p-heterogeneity > 0.5). Our data confirm suggested associations between DM1 and skin neoplasms with the highest risk seen for BCC. Patients are advised to minimize ultraviolet light exposure and seek medical advice for suspicious lesions.

Does an expressed sequence tag (EST) library of Salsola iberica (tumbleweed) help to understand plant responses to environmental stresses?

Weeds play an important role in agriculture and molecular techniques are useful to help understand traits that contribute to weediness and weeds' interactions with the environment. A total of 377 expressed sequence tags (ESTs) from a modest library were arranged into 227 unique fragments and 61 contigs, which consisted of two or more ESTs. From blastx results, we mapped and annotated unigenes using the gene ontology vocabulary according to biological process, cellular component and molecular function. These were then compared to a reference set of Arabidopsis thaliana sequences for statistically significant over- or underrepresented genes. The sequences were also compared against multiple protein databases for similarity of functional domains. Overall, the S. iberica sequences showed high similarity to response to stress, which included salt-induced proteins, betaine aldehydehyde dehydrogenase and calcium binding proteins. Only a modest number of transcripts were sequenced; however, the results presented here demonstrate the metabolic versatility of S. iberica in sub-optimal conditions that are likely to contribute to its cosmopolitan distribution. Here we propose that an EST library of an economically important weed species could be used to understand the weed's interactions with the environment.