Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology

Barkovskaya, M. Sh.; Bogomolov, Anton; Knauer, Nadezhda; Rubtsov, N. B.; Козлов, В. А.

doi:10.1142/s0219720016500414

Cited by 7 publications

(9 citation statements)

References 31 publications

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“…Качество полученных препаратов контролировали на фазово-контрастном микроскопе. Препараты с наилучшим распластыванием метафазных пластинок выдерживали в течение 1 ч при температуре 60 °С и подвергали реакции Q-FISH с использованием зонда PNA (Peptide Nucleic Acid), меченного Cy3 (CCCTAA)3 (Eurogenetec Ltd., Бельгия) в подобранных нами ранее условиях [21].…”

Section: материалы и методыunclassified

“…Флуоресцентный микроскоп Axioscop 40 (ZEISS, Германия) применялся для микроскопии и регистрации изображения в соответствии с ранее описанной методикой [21]. C целью нормирования осветительной системы использовали флуоресцентные бусы (Fluospheres 0,2 мкм, Life Technologies, США) с оранжевым спектром флуоресценции (540/560 нм), которые предварительно наносились на предметное стекло и заключались в антифýйд.…”

Section: материалы и методыunclassified

“…Анализ изображений. Цифровая обработка изображений проводилась в специально созданной программе MeTeLen с учетом времени ýкспозиции, процедур нормировки и выравнивания, как было описано ранее [21]…”

Section: материалы и методыunclassified

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Telomere length distribution on individual chromosome arms in patients with bronchial asthma

et al. 2019

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Objective. The purpose of this study was to evaluate the length of telomeres in the arms of individual chromosomes in patients with bronchial asthma (BA).Materials and methods. The study included patients with BA (n = 10, the mean age (44 ± 8.2) years) and healthy donors (n = 10, the mean age (44 ± 8.4) years). Metaphase spreads obtained from peripheral blood mononuclear cells were used. At the time of sampling BA patients received treatment at the Clinic of Immunopathology, Novosibirsk. BA was diagnosed by physicians according to GINA-2016. For measurement of telomere length on individual chromosome arms we used quantitative fluorescent in situ hybridization with a PNA-probe specific for telomeres. We used inverted DAPI banding for chromosome identification (according to ISCN-2013). For each individual 5 metaphase cells were analyzed. We applied the newly developed MeTeLen software to estimate the telomere repeats quantity (http:// www.bionet.nsc.ru/en/development/application-development/development-of-a-computer/metelen.html) in metaphase images. For enhanced image analysis compared with the previously developed programs, we included estimation of background signal and correction of defects of the optical system.Results. Comparing of telomere length show, that telomeres in the certain chromosome arms (4q, 5q, 9p, 10 q, 11p, 13p, 15q, 18q, 19q) in BA are significantly shorter than in corresponding group of donors (p < 0.05, Mann – Whitney U-test). For both studied groups we also evaluated telomere sequences shortened and elongated relative to the average telomere length in the group (p < 0.05, Wilcoxon-signed-runk test). The following differences and similarities between the telomere profiles of patients and donors were determined: the telomere sequences 4p, 6q, 8p were elongated and 2q, 9q, 11p, 15q were shortened relative to the average telomere length in BA patients. Moreover, this telomere sequences did not differ from the average telomere length in the group of donors. At the same time, the telomere sequences 12p, 16p, 17p, 19p were significantly shorter, and 3p was longer than the average telomere length in both groups.Conclusions. We guess, that the observed significant shortening of telomere length on individual chromosome arms in BA, as compared to donors, is relevant in pathogenesis of this disorder. The revealed features of telomere profile of patients with BA may be a result of different telomere length maintenance mechanisms and may influence to the development of asthma that needs further study.

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Telomere length distribution on individual chromosome arms in patients with bronchial asthma

et al. 2019

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“…9,[13][14][15] Recently several methods including quantitative polymerase chain reaction (Q-PCR), quantitative fluorescence in situ hybridization (Q-FISH), flow fluorescence in situ hybridization, and single telomere length analysis were used for measuring telomere length. 16,17 In this study, we assessed gene mutation and telomere length as prognostic role in patient with MDS with integrating this variable into a revised IPSS. And, we aim to find additional factors to be of additive prognostic value in MDS.…”

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confidence: 99%

Telomere integrated scoring system of myelodysplastic syndrome

Park

Shin

et al. 2023

Clinical Laboratory Analysis

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Introduction:Recently, multigene target sequencing is widely performed for the purpose of prognostic prediction and application of targeted therapy. Here, we proposed a new scoring system that encompasses gene variations, telomere length, and Revised International Prognostic Scoring System (IPSS-R) together in Asian myelodysplastic syndrome. Methods:We developed a new scoring model of these variables: age ≥ 65 years + IPSS-R score + ASXL1 mutation + TP53 mutation + Telomere length (<5.37). According to this new scoring system, patients were divided into four groups: very good score cutoff (≤3.0), good (3.0-4.5), poor (4.5-7.0), and very poor (>7.0). Results:The median OS was 170.1, 100.4, 46.0, and 12.0 months for very good, good, poor, and very poor, retrospectively (p < 0.001). Meanwhile, according to the conventional IPSS-R scoring system, the median OS was 141.3, 50.2, 93.0, 36.0, and 16.2 months for very low, low, intermediate, high, and very high, retrospectively (p < 0.001). Conclusions:The newly developed model incorporating molecular variations and TL yielded more clear separations of the survival curves. By adding the presence of gene mutation and telomere length to the existing IPSS-R, its predictive ability can be further improved in myelodysplastic syndrome.

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“…One such example is the application of an open-source Java program, Telometric, which has been reported to provide an overestimation of mean TL when processing agarose gel images (Grant et al, 2001; Haussmann et al, 2011). MeTelenisa freeware software with special options like correction of irregular light effects and elimination of background fluorescence in Q-FISH images and has been used widely (Barkovskaya et al, 2016). On the other hand, Computel estimates mean TL from the whole genome based on sequencing data (Nersisyan and Arakelyan, 2015).…”

Section: Introductionmentioning

confidence: 99%

Developing BIOTEL: A Semi-Automated Spreadsheet for Estimating Telomere Length and Biological Age

et al. 2019

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Introduction: Telomere length (TL) is causally related to aging and several age-related diseases. Specifically, the abundance of short telomeres and the rate of telomere shortening are strong determinants of cell homeostasis. Thus, tools for analyzing and manipulating TL data can vastly improve research focused on aging. Aim: In this study, we developed a semi-automated worksheet, BIOTEL, to generate individual and group TL statistics and provide a crude estimation of biological age. Results: Data from the Telomere Length Database Project (TLDP) were implemented to the spreadsheet to produce TL statistics. 150 participants were included, and their age was from 21 to 82 years, and the sex distribution ratio was 52.3%: 47.7% (male: female). Initially, we analyzed the fluorescence intensities of telomeres that were measured on metaphase spread leukocytes using three-dimensional (3D) quantitative-fluorescent in situ hybridization (Q-FISH) procedures (3D DNA FISH) with a (C3TA2)3 peptide nucleic acid (PNA) probe. Raw data of fluorescence intensities, demographic data and medical records from the participants were imported into the worksheet. Basic statistical analyses of TL data were provided through BIOTEL, including TL percentiles, specialized charts for TL distribution including the percentage of critically short telomeres (< 3,000 kilobases), individual telomere profiles, and graphs of biological age vs. chronological age. Conclusion: BIOTEL ver. 2.4 is a functional semi-automated worksheet that calculates a wide range of TL statistics, thus a useful tool with applications in research of telomeres and biological age estimation.

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Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology

Cited by 7 publications

References 31 publications

Telomere length distribution on individual chromosome arms in patients with bronchial asthma

Telomere length distribution on individual chromosome arms in patients with bronchial asthma

Telomere integrated scoring system of myelodysplastic syndrome

Developing BIOTEL: A Semi-Automated Spreadsheet for Estimating Telomere Length and Biological Age

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