Monitoring of telomere dynamics in peripheral blood leukocytes in relation to colorectal cancer patients' outcomes.

We investigated the possible associations between leukocyte telomere length, therapy outcomes, and clinicopathological features in patients with colorectal cancer. Additionally, telomerase reverse transcriptase (TERT) expression was evaluated. Telomere length was measured using singleplex qPCR in 478 consecutive leukocyte DNA samples from 198 patients. Blood was drawn at diagnosis prior to any therapy and then at 6-month intervals for 18 months. Following diagnosis, the telomeres gradually shortened during the course of the treatment regardless of the patient's age. The most pronounced decrease was observed 12 months after the diagnosis (p < 0.0001). Based on tumor localization, the decrease in telomere length one year after the diagnosis followed different trajectories (p = 0.03). In patients treated with adjuvant therapy, telomere length correlated with the time elapsed after completion of therapy (p = 0.03). TERT expression did not correlate with the telomere length; however, it was higher in women than men (1.35-fold, 95% CI 1.11-1.65, p = 0.003) and in smokers than non-smokers (1.27-fold, 95% CI 1.01-1.61, p = 0.04). Leukocyte telomere length declines naturally during aging, but the accelerated shortening observed in our patients was age-independent. Telomere length manifestly reflected chemotherapy impact and could be linked to therapy toxicity.

Improving structural variant clustering to reduce the negative effect of the breakpoint uncertainty problem.

BACKGROUND: Structural variants (SVs) represent an important source of genetic variation. One of the most critical problems in their detection is breakpoint uncertainty associated with the inability to determine their exact genomic position. Breakpoint uncertainty is a characteristic issue of structural variants detected via short-read sequencing methods and complicates subsequent population analyses. The commonly used heuristic strategy reduces this issue by clustering/merging nearby structural variants of the same type before the data from individual samples are merged. RESULTS: We compared the two most used dissimilarity measures for SV clustering in terms of Mendelian inheritance errors (MIE), kinship prediction, and deviation from Hardy-Weinberg equilibrium. We analyzed the occurrence of Mendelian-inconsistent SV clusters that can be collapsed into one Mendelian-consistent SV as a new measure of dataset consistency. We also developed a new method based on constrained clustering that explicitly identifies these types of clusters. CONCLUSIONS: We found that the dissimilarity measure based on the distance between SVs breakpoints produces slightly better results than the measure based on SVs overlap. This difference is evident in trivial and corrected clustering strategy, but not in constrained clustering strategy. However, constrained clustering strategy provided the best results in all aspects, regardless of the dissimilarity measure used.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study.

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Cell-free DNA from human plasma and serum differs in content of telomeric sequences and its ability to promote immune response.

Circulating cell-free DNA (cfDNA) may be involved in immune response regulation. We studied the variations in abundance of telomeric sequences in plasma and serum in young healthy volunteers and the ability of cfDNA contained in these samples to co-activate the TNF-α m RNA expression in monocytes. We performed qPCR to determine relative telomere length (T/S ratios) in plasma, serum and whole blood of 36 volunteers. Using paired samples of plasma and serum and DNase treatment, we analysed the contribution of cfDNA to the co-activation of TNF-α mRNA expression in THP1 monocytic cell line. We found significant differences between paired plasma and serum samples in relative T/S ratios (median 1.38 ± 1.1 vs. 0.86 ± 0.25, respectively) and in total amounts of cfDNA and in estimated total amounts of telomeres which were significantly higher in serum than in plasma. TNF-α mRNA expression in THP1 cells increased significantly after DNase treatment of all samples used for stimulation. The highest TNF-α mRNA expressions were observed after stimulation with DNase treated serum samples. Our results suggest that the different content of telomeric sequences in plasma and serum may contribute to the tuning of immune response. Further studies of this interesting phenomenon are needed.

Relative amount of telomeric sequences in terminal villi does not differ between normal term placentas and placentas from patients with well-controlled type 1 diabetes mellitus.

We applied qPCR to compare relative telomere length in terminal villi microdissected from term control placentas and placentas of patients suffering from type 1 diabetes. Significant differences were not found in the relative T/S ratios between placental groups or between the diabetic placentas affected and those not affected with chorangiosis. We hypothesize that there is no relationship between decreased placental proliferative ability in maternal diabetes type 1 and telomere shortening.