Measuring vertebrate telomeres: applications and limitations

Telomeres are short tandem repeated sequences of DNA found at the ends of eukaryotic chromosomes that function in stabilizing chromosomal end integrity. In vivo studies of somatic tissue of mammals and birds have shown a correlation between telomere length and organismal age within species, and correlations between telomere shortening rate and lifespan among species. This result presents the tantalizing possibility that telomere length could be used to provide much needed information on age, ageing and survival in natural populations where longitudinal studies are lacking. Here we review methods available for measuring telomere length and discuss the potential uses and limitations of telomeres as age and ageing estimators in the fields of vertebrate ecology, evolution and conservation

Shallow WGS of individual CTCs identifies actionable targets for informing treatment decisions in metastatic breast cancer

Background We report copy-number profiling by low-pass WGS (LP-WGS) in individual circulating tumour cells (CTCs) for guiding treatment in patients with metastatic breast cancer (MBC), comparing CTC results with mutations detected in circulating tumour DNA (ctDNA) in the same blood samples. Methods Across 10 patients with MBC who were progressing at the time of blood sampling and that had >20 CTCs detected by CellSearch®, 63 single cells (50 CTCs and 13 WBCs) and 16 cell pools (8 CTC pools and 8 WBC pools) were recovered from peripheral blood by CellSearch®/DEPArray™ and sequenced with Ampli1 LowPass technology (Menarini Silicon Biosystems). Copy-number aberrations were identified using the MSBiosuite software platform, and results were compared with mutations detected in matched plasma cfDNA analysed by targeted next-generation sequencing using the Oncomine™ Breast cfDNA Assay (Thermo Fisher). Results LP-WGS data demonstrated copy-number gains/losses in individual CTCs in regions including FGFR1, JAK2 and CDK6 in five patients, ERBB2 amplification in two HER2-negative patients and BRCA loss in two patients. Seven of eight matched plasmas also had mutations in ctDNA in PIK3CA, TP53, ESR1 and KRAS genes with mutant allele frequencies (MAF) ranging from 0.05 to 33.11%. Combining results from paired CTCs and ctDNA, clinically actionable targets were identified in all ten patients. Conclusion This combined analysis of CTCs and ctDNA may offer a new approach for monitoring of disease progression and to direct therapy in patients with advanced MBC, at a time when they are coming towards the end of other treatment options

A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation

Breast cancer (BC) is a common cancer in women worldwide. Despite advances in treatment, up to 30% of women eventually relapse and die of metastatic breast cancer. Liquid biopsy analysis of circulating cell-free DNA fragments in the patients’ blood can monitor clonality and evolving mutations as a surrogate for tumour biopsy. Next generation sequencing platforms and digital droplet PCR can be used to profile circulating tumour DNA from liquid biopsies; however, they are expensive and time consuming for clinical use. Here, we report a novel strategy with proof-of-concept data that supports the usage of loop-mediated isothermal amplification (LAMP) to detect PIK3CA c.3140 A > G (H1047R), a prevalent BC missense mutation that is attributed to BC tumour growth. Allele-specific primers were designed and optimized to detect the p.H1047R variant following the USS-sbLAMP method. The assay was developed with synthetic DNA templates and validated with DNA from two breast cancer cell-lines and two patient tumour tissue samples through a qPCR instrument and finally piloted on an ISFET enabled microchip. This work sets a foundation for BC mutational profiling on a Lab-on-Chip device, to help the early detection of patient relapse and to monitor efficacy of systemic therapies for personalised cancer patient management

Extreme longevity variants at the FOXO3 locus may moderate FOXO3 isoform levels

This is the final version. Available on open access from Springer via the DOI in this recordThe rs2802292, rs2764264 and rs13217795 variants of FOXO3 have been associated with extreme longevity in multiple human populations, but the mechanisms underpinning this remain unclear. We aimed to characterise potential effects of longevity-associated variation on the expression and mRNA processing of the FOXO3 gene. We performed a comprehensive assessment of FOXO3 isoform usage across a wide variety of human tissues and carried out a bioinformatic analysis of the potential for longevity-associated variants to disrupt regulatory regions involved in isoform choice. We then related the expression of full length and 5' truncated FOXO3 isoforms to rs13217795 genotype in peripheral blood and skeletal muscle from individuals of different rs13217795 genotypes. FOXO3 isoforms displayed considerable tissue specificity. We determined that rs13231195 and its tightly aligned proxy variant rs9400239 may lie in regulatory regions involved in isoform choice. The longevity allele at rs13217795 was associated with increased levels of full length FOXO3 isoforms in peripheral blood and a decrease in truncated FOXO3 isoforms in skeletal muscle RNA. We suggest that the longevity effect of FOXO3 SNPs may in part derive from a shift in isoform usage in skeletal muscle away from the production of 5' truncated FOXO3 isoforms lacking a complete forkhead DNA binding domain, which may have compromised functionality.National Institute for Health Research (NIHR)Kuakini Medical Center, the US National Institutes of HealthNational Heart, Lung, and Blood InstituteUniversity of Exete

Satellite Cells Senescence in Limb Muscle of Severe Patients with COPD

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec, Canada Rationale: The maintenance of peripheral muscle mass may be compromised in chronic obstructive pulmonary disease (COPD) due to premature cellular senescence and exhaustion of the regenerative potential of the muscles. Methods: Vastus lateralis biopsies were obtained from patients with COPD (n = 16) and healthy subjects (n = 7). Satellite cell number and the proportion of central nuclei, as a marker of muscle regenerative events, were assessed on cryosections. Telomere lengths, used as a marker of cellular senescence, were determined using Southern blot analyses. Results: Central nuclei proportion was significantly higher in patients with COPD with a preserved muscle mass compared to controls and patients with COPD with muscle atrophy (p,0.001). In COPD, maximal telomere length was significantly decreased compared to controls (p,0.05). Similarly, minimal telomere length was significantly reduced in GOLD III–IV patients with muscle atrophy compared to controls (p,0.005). Minimal, mean and maximum telomere lengths correlated with mid-thigh muscle cross-sectional area (MTCSA) (R = 0.523, p = 0.005; R = 0.435, p = 0.019 and R = 0.491, p = 0.009, respectively). Conclusions: Evidence of increased regenerative events was seen in GOLD III–IV patients with preserved muscle mass. Shortening of telomeres in GOLD III–IV patients with muscle atrophy is consistent with an increased number of senescen

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA

<p>Abstract</p> <p>Background</p> <p>The epidemiology of <it>E. ruminantium </it>infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia.</p> <p>Methods</p> <p>We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of <it>E. ruminantium </it>infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting <it>E. ruminantium </it>infection was also assessed.</p> <p>Results</p> <p>The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable <it>A. variegatum </it>infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher <it>E. ruminantium </it>prevalence in the animals with increasing age and both the Spearman's rank test (<it>r</it>_{<it>s </it>}= -0.1512; P = 0.003) and <it>kappa </it>statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays.</p> <p>Conclusion</p> <p>The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.</p

Lung fibroblasts from patients with emphysema show markers of senescence in vitro

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated β-galactosidase (SA-β-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-β-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor ≥ 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length

Examination of NRCAM, LRRN3, KIAA0716, and LAMB1 as autism candidate genes

BACKGROUND: A substantial body of research supports a genetic involvement in autism. Furthermore, results from various genomic screens implicate a region on chromosome 7q31 as harboring an autism susceptibility variant. We previously narrowed this 34 cM region to a 3 cM critical region (located between D7S496 and D7S2418) using the Collaborative Linkage Study of Autism (CLSA) chromosome 7 linked families. This interval encompasses about 4.5 Mb of genomic DNA and encodes over fifty known and predicted genes. Four candidate genes (NRCAM, LRRN3, KIAA0716, and LAMB1) in this region were chosen for examination based on their proximity to the marker most consistently cosegregating with autism in these families (D7S1817), their tissue expression patterns, and likely biological relevance to autism. METHODS: Thirty-six intronic and exonic single nucleotide polymorphisms (SNPs) and one microsatellite marker within and around these four candidate genes were genotyped in 30 chromosome 7q31 linked families. Multiple SNPs were used to provide as complete coverage as possible since linkage disequilibrium can vary dramatically across even very short distances within a gene. Analyses of these data used the Pedigree Disequilibrium Test for single markers and a multilocus likelihood ratio test. RESULTS: As expected, linkage disequilibrium occurred within each of these genes but we did not observe significant LD across genes. None of the polymorphisms in NRCAM, LRRN3, or KIAA0716 gave p < 0.05 suggesting that none of these genes is associated with autism susceptibility in this subset of chromosome 7-linked families. However, with LAMB1, the allelic association analysis revealed suggestive evidence for a positive association, including one individual SNP (p = 0.02) and three separate two-SNP haplotypes across the gene (p = 0.007, 0.012, and 0.012). CONCLUSIONS: NRCAM, LRRN3, KIAA0716 are unlikely to be involved in autism. There is some evidence that variation in or near the LAMB1 gene may be involved in autism