Publications by authors named "Rosa Ana Risques"

44 Publications

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195;

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
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http://dx.doi.org/10.1073/pnas.2023373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346827PMC
August 2021

Characterizing TP53 mutations in ovarian carcinomas with and without concurrent BRCA1 or BRCA2 mutations.

Gynecol Oncol 2021 03 26;160(3):786-792. Epub 2020 Dec 26.

Department of Obstetrics and Gynecology, University of Washington, Seattle, WA, USA.

Objectives: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes.

Methods: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance.

Results: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome.

Conclusions: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type.
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http://dx.doi.org/10.1016/j.ygyno.2020.12.007DOI Listing
March 2021

Telomere-to-telomere assembly of a complete human X chromosome.

Nature 2020 09 14;585(7823):79-84. Epub 2020 Jul 14.

Arima Genomics, San Diego, CA, USA.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist. Here we present a human genome assembly that surpasses the continuity of GRCh38, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
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http://dx.doi.org/10.1038/s41586-020-2547-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484160PMC
September 2020

Characterization of TP53 mutations in Pap test DNA of women with and without serous ovarian carcinoma.

Gynecol Oncol 2020 02 12;156(2):407-414. Epub 2019 Dec 12.

Department of Pathology, University of Washington, Seattle, WA 98195, USA. Electronic address:

Objective: Pap tests hold promise as a molecular diagnostic for serous ovarian cancer, but previous studies reported limited sensitivity. Furthermore, the presence of somatic mutations in normal tissue is increasingly recognized as a challenge to the specificity of mutation-based cancer diagnostics. We applied an ultra-deep sequencing method with the goal of improving sensitivity and characterizing the landscape of low-frequency somatic TP53 mutations in Pap tests.

Methods: We used CRISPR-DS to deeply sequence (mean Duplex depth ~3000×) the TP53 gene in 30 Pap tests from 21 women without cancer and 9 women with serous ovarian carcinoma with known TP53 driver mutations. Mutations were annotated and compared to those in the TP53 cancer database.

Results: The tumor-derived mutation was identified in 3 of 8 Pap tests from women with ovarian cancer and intact tubes. In addition, 221 low-frequency (≲0.001) exonic TP53 mutations were identified in Pap tests from women with ovarian cancer (94 mutations) and without ovarian cancer (127 mutations). Many of these mutations resembled TP53 mutations found in cancer: they impaired protein activity, were predicted to be pathogenic, and clustered in exons 5 to 8 and hotspot codons. Cancer-like mutations were identified in all women but at higher frequency in women with ovarian cancer.

Conclusions: Pap tests have low sensitivity for ovarian cancer detection and carry abundant low-frequency TP53 mutations. These mutations are more frequently pathogenic in women with ovarian cancer. Determining whether low-frequency TP53 mutations in normal gynecologic tissues are associated with an increased cancer risk warrants further study.
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http://dx.doi.org/10.1016/j.ygyno.2019.11.124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018621PMC
February 2020

Cancer-Associated Mutations but No Cancer: Insights into the Early Steps of Carcinogenesis and Implications for Early Cancer Detection.

Trends Cancer 2019 09 22;5(9):531-540. Epub 2019 Aug 22.

Department of Pathology, University of Washington, Seattle, WA, USA. Electronic address:

Cancer is a disease of aging fueled by the accumulation of somatic mutations. While mutations in tumors are well characterized, little is known about the early mutational processes that initiate tumorigenesis. Recent advances in next-generation sequencing (NGS) have enabled the detection of mutations in normal tissue, revealing an unanticipated high level of age-related somatic mutations affecting most individuals and tissues. Surprisingly, many of these mutations are similar to mutations commonly found in tumors, suggesting an ongoing process of positive selection and clonal expansion akin to what occurs in cancer, but within normal tissue. Here we discuss some of the most important biological and clinical implications of these novel findings, with a special focus on their impact for cancer detection and prediction.
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http://dx.doi.org/10.1016/j.trecan.2019.07.007DOI Listing
September 2019

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue over a Century of Human Lifespan.

Cell Rep 2019 07;28(1):132-144.e3

Department of Pathology, University of Washington, Seattle, WA 98195, USA. Electronic address:

High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low-frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging, and great care must be taken to distinguish tumor-derived from age-associated mutations in high-sensitivity clinical cancer diagnostics.
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http://dx.doi.org/10.1016/j.celrep.2019.05.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639023PMC
July 2019

Mitochondrial DNA Mutations are Associated with Ulcerative Colitis Preneoplasia but Tend to be Negatively Selected in Cancer.

Mol Cancer Res 2019 02 16;17(2):488-498. Epub 2018 Nov 16.

Department of Pathology, University of Washington, Seattle, Washington.

The role of mitochondrial DNA (mtDNA) mutations in cancer remains controversial. Ulcerative colitis is an inflammatory bowel disease that increases the risk of colorectal cancer and involves mitochondrial dysfunction, making it an ideal model to study the role of mtDNA in tumorigenesis. Our goal was to comprehensively characterize mtDNA mutations in ulcerative colitis tumorigenesis using Duplex Sequencing, an ultra-accurate next-generation sequencing method. We analyzed 46 colon biopsies from non-ulcerative colitis control patients and ulcerative colitis patients with and without cancer, including biopsies at all stages of dysplastic progression. mtDNA was sequenced at a median depth of 1,364x. Mutations were classified by mutant allele frequency: clonal > 0.95, subclonal 0.01-0.95, and very low frequency (VLF) < 0.01. We identified 208 clonal and subclonal mutations and 56,764 VLF mutations. Mutations were randomly distributed across the mitochondrial genome. Clonal and subclonal mutations increased in number and pathogenicity in early dysplasia, but decreased in number and pathogenicity in cancer. Most clonal, subclonal, and VLF mutations were C>T transitions in the heavy strand of mtDNA, which likely arise from DNA replication errors. A subset of VLF mutations were C>A transversions, which are probably due to oxidative damage. VLF transitions and indels were less abundant in the non-D-loop region and decreased with progression. Our results indicate that mtDNA mutations are frequent in ulcerative colitis preneoplasia but negatively selected in cancers. IMPLICATIONS: While mtDNA mutations might contribute to early ulcerative colitis tumorigenesis, they appear to be selected against in cancer, suggesting that functional mitochondria might be required for malignant transformation in ulcerative colitis.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481604PMC
February 2019

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Genome Res 2018 10 19;28(10):1589-1599. Epub 2018 Sep 19.

Department of Pathology, University of Washington, Seattle, Washington 98195, USA.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.
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http://dx.doi.org/10.1101/gr.235291.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169890PMC
October 2018

Seshat: A Web service for accurate annotation, validation, and analysis of TP53 variants generated by conventional and next-generation sequencing.

Hum Mutat 2018 07 17;39(7):925-933. Epub 2018 May 17.

Sorbonne Université, UPMC Univ Paris, Paris, France.

Accurate annotation of genomic variants in human diseases is essential to allow personalized medicine. Assessment of somatic and germline TP53 alterations has now reached the clinic and is required in several circumstances such as the identification of the most effective cancer therapy for patients with chronic lymphocytic leukemia (CLL). Here, we present Seshat, a Web service for annotating TP53 information derived from sequencing data. A flexible framework allows the use of standard file formats such as Mutation Annotation Format (MAF) or Variant Call Format (VCF), as well as common TXT files. Seshat performs accurate variant annotations using the Human Genome Variation Society (HGVS) nomenclature and the stable TP53 genomic reference provided by the Locus Reference Genomic (LRG). In addition, using the 2017 release of the UMD_TP53 database, Seshat provides multiple statistical information for each TP53 variant including database frequency, functional activity, or pathogenicity. The information is delivered in standardized output tables that minimize errors and facilitate comparison of mutational data across studies. Seshat is a beneficial tool to interpret the ever-growing TP53 sequencing data generated by multiple sequencing platforms and it is freely available via the TP53 Website, http://p53.fr or directly at http://vps338341.ovh.net/.
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http://dx.doi.org/10.1002/humu.23543DOI Listing
July 2018

All's well that ends well: why large species have short telomeres.

Philos Trans R Soc Lond B Biol Sci 2018 03;373(1741)

Department of Pathology, University of Washington, Seattle, WA 98195, USA.

Among mammal species, almost all life-history traits are strongly size dependent. This size dependence even occurs at a molecular level. For example, both telomere length and telomerase expression show a size-dependent threshold. With some exceptions, species smaller than approximately 2 kg express telomerase, while species larger than that do not. Among species greater than approximately 5 kg, telomeres tend to be short-less than 25 kb-while among smaller species, some species have short and some have long telomeres. Here, we present a model to explore the role of body size-dependent trade-offs in shaping this threshold. We assume that selection favours short telomeres as a mechanism to protect against cancer. At the same time, selection favours long telomeres as a protective mechanism against DNA damage and replicative senescence. The relative importance of these two selective forces will depend on underlying intrinsic mortality and risk of cancer, both of which are size-dependent. Results from this model suggest that a cost-benefit model for the evolution of telomere length could explain phylogenetic patterns observed within the Class Mammalia. In addition, the model suggests a general conceptual framework to think about the role that body size plays in the evolution of tumour suppressor mechanisms.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.
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http://dx.doi.org/10.1098/rstb.2016.0448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5784068PMC
March 2018

Aging and the rise of somatic cancer-associated mutations in normal tissues.

PLoS Genet 2018 01 4;14(1):e1007108. Epub 2018 Jan 4.

Department of Pathology, University of Washington, Seattle, Washington, United States of America.

DNA mutations are inevitable. Despite proficient DNA repair mechanisms, somatic cells accumulate mutations during development and aging, generating cells with different genotypes within the same individual, a phenomenon known as somatic mosaicism. While the existence of somatic mosaicism has long been recognized, in the last five years, advances in sequencing have provided unprecedented resolution to characterize the extent and nature of somatic genetic variation. Collectively, these new studies are revealing a previously uncharacterized aging phenotype: the accumulation of clones with cancer driver mutations. Here, we summarize the most recent findings, which converge in the novel notion that cancer-associated mutations are prevalent in normal tissue and accumulate with aging.
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http://dx.doi.org/10.1371/journal.pgen.1007108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754046PMC
January 2018

Precancer in ulcerative colitis: the role of the field effect and its clinical implications.

Carcinogenesis 2018 01;39(1):11-20

Division of Gasteroenterology, Department of Medicine, University of Washington, Seattle, WA, USA.

Cumulative evidence indicates that a significant proportion of cancer evolution may occur before the development of histological abnormalities. While recent improvements in DNA sequencing technology have begun to reveal the presence of these early preneoplastic clones, the concept of 'premalignant field' was already introduced by Slaughter more than half a century ago. Also referred to as 'field effect', 'field defect' or 'field cancerization', these terms describe the phenomenon by which molecular alterations develop in normal-appearing tissue and expand to form premalignant patches with the potential to progress to dysplasia and cancer. Field effects have been well-characterized in ulcerative colitis, an inflammatory bowel disease that increases the risk of colorectal cancer. The study of the molecular alterations that define these fields is informative of mechanisms of tumor initiation and progression and has provided potential targets for early cancer detection. Herein, we summarize the current knowledge about the molecular alterations that comprise the field effect in ulcerative colitis and the clinical utility of these fields for cancer screening and prevention.
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http://dx.doi.org/10.1093/carcin/bgx117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6248676PMC
January 2018

Magellanic penguin telomeres do not shorten with age with increased reproductive effort, investment, and basal corticosterone.

Ecol Evol 2017 08 15;7(15):5682-5691. Epub 2017 Jun 15.

Center for Ecosystem Sentinals Department of BiologyUniversity of Washington Seattle WA USA.

All species should invest in systems that enhance longevity; however, a fundamental adult life-history trade-off exists between the metabolic resources allocated to maintenance and those allocated to reproduction. Long-lived species will invest more in reproduction than in somatic maintenance as they age. We investigated this trade-off by analyzing correlations among telomere length, reproductive effort and output, and basal corticosterone in Magellanic penguins (). Telomeres shorten with age in most species studied to date, and may affect adult survival. High basal corticosterone is indicative of stressful conditions. Corticosterone, and stress, has been linked to telomere shortening in other species. Magellanic penguins are a particularly good model organism for this question as they are an unusually long-lived species, exceeding their mass-adjusted predicted lifespan by 26%. Contrary to our hypothesis, we found adults aged 5 years to over 24 years of age had similar telomere lengths. Telomeres of adults did not shorten over a 3-year period, regardless of the age of the individual. Neither telomere length, nor the rate at which the telomeres changed over these 3 years, correlated with breeding frequency or investment. Older females also produced larger volume clutches until approximately 15 years old and larger eggs produced heavier fledglings. Furthermore, reproductive success () is maintained as females aged. Basal corticosterone, however, was not correlated with telomere length in adults and suggests that low basal corticosterone may play a role in the telomere maintenance we observed. Basal corticosterone also declined during the breeding season and was positively correlated with the age of adult penguins. This higher basal corticosterone in older individuals, and consistent reproductive success, supports the prediction that Magellanic penguins invest more in reproduction as they age. Our results demonstrate that telomere maintenance may be a component of longevity even with increased reproductive effort, investment, and basal corticosterone.
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http://dx.doi.org/10.1002/ece3.3128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552965PMC
August 2017

Telomeres shorten and then lengthen before fledging in Magellanic penguins ().

Aging (Albany NY) 2017 02;9(2):487-493

Department of Biology, University of Washington, Seattle, WA 98195, USA.

For all species, finite metabolic resources must be allocated toward three competing systems: maintenance, reproduction, and growth. Telomeres, the nucleoprotein tips of chromosomes, which shorten with age in most species, are correlated with increased survival. Chick growth is energetically costly and is associated with telomere shortening in most species. To assess the change in telomeres in penguin chicks, we quantified change in telomere length of wild known-age Magellanic penguin () chicks every 15 days during the species' growth period, from hatching to 60 days-of-age. Magellanic penguins continue to grow after fledging so we also sampled a set of 1-year-old juvenile penguins, and adults aged 5 years. Telomeres were significantly shorter on day 15 than on hatch day but returned to their initial length by 30 days old and remained at that length through 60 days of age. The length of telomeres of newly hatched chicks, chicks aged 30, 45 and 60 days, juveniles, and adults aged 5 years were similar. Chicks that fledged and those that died had similar telomere lengths. We show that while telomeres shorten during growth, Magellanic penguins elongate telomeres to their length at hatch, which may increase adult life span and reproductive opportunities.
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http://dx.doi.org/10.18632/aging.101172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361676PMC
February 2017

Werner syndrome through the lens of tissue and tumour genomics.

Sci Rep 2016 08 25;6:32038. Epub 2016 Aug 25.

Department of Pathology, University of Washington, Seattle, WA USA.

Werner syndrome (WS) is the canonical adult human progeroid ('premature aging') syndrome. Patients with this autosomal recessive Mendelian disorder display constitutional genomic instability and an elevated risk of important age-associated diseases including cancer. Remarkably few analyses of WS patient tissue and tumors have been performed to provide insight into WS disease pathogenesis or the high risk of neoplasia. We used autopsy tissue from four mutation-typed WS patients to characterize pathologic and genomic features of WS, and to determine genomic features of three neoplasms arising in two of these patients. The results of these analyses provide new information on WS pathology and genomics; provide a first genomic characterization of neoplasms arising in WS; and provide new histopathologic and genomic data to test several popular models of WS disease pathogenesis.
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http://dx.doi.org/10.1038/srep32038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997333PMC
August 2016

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Proc Natl Acad Sci U S A 2016 May 5;113(21):6005-10. Epub 2016 May 5.

Department of Pathology, University of Washington, Seattle, WA 98195;

Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.
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http://dx.doi.org/10.1073/pnas.1601311113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889384PMC
May 2016

Predicting Survival from Telomere Length versus Conventional Predictors: A Multinational Population-Based Cohort Study.

PLoS One 2016 6;11(4):e0152486. Epub 2016 Apr 6.

Center for Population and Health, Georgetown University, Washington, District of Columbia, United States of America.

Telomere length has generated substantial interest as a potential predictor of aging-related diseases and mortality. Some studies have reported significant associations, but few have tested its ability to discriminate between decedents and survivors compared with a broad range of well-established predictors that include both biomarkers and commonly collected self-reported data. Our aim here was to quantify the prognostic value of leukocyte telomere length relative to age, sex, and 19 other variables for predicting five-year mortality among older persons in three countries. We used data from nationally representative surveys in Costa Rica (N = 923, aged 61+), Taiwan (N = 976, aged 54+), and the U.S. (N = 2672, aged 60+). Our study used a prospective cohort design with all-cause mortality during five years post-exam as the outcome. We fit Cox hazards models separately by country, and assessed the discriminatory ability of each predictor. Age was, by far, the single best predictor of all-cause mortality, whereas leukocyte telomere length was only somewhat better than random chance in terms of discriminating between decedents and survivors. After adjustment for age and sex, telomere length ranked between 15th and 17th (out of 20), and its incremental contribution was small; nine self-reported variables (e.g., mobility, global self-assessed health status, limitations with activities of daily living, smoking status), a cognitive assessment, and three biological markers (C-reactive protein, serum creatinine, and glycosylated hemoglobin) were more powerful predictors of mortality in all three countries. Results were similar for cause-specific models (i.e., mortality from cardiovascular disease, cancer, and all other causes combined). Leukocyte telomere length had a statistically discernible, but weak, association with mortality, but it did not predict survival as well as age or many other self-reported variables. Although telomere length may eventually help scientists understand aging, more powerful and more easily obtained tools are available for predicting survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152486PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822878PMC
August 2016

Obesity and inflammation markers in relation to leukocyte telomere length in a cross-sectional study of persons with Barrett's esophagus.

BMC Obes 2015 10;2:32. Epub 2015 Sep 10.

Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA USA ; Department of Epidemiology, University of Washington, Seattle, WA USA.

Background: Telomere shortening is associated with increasing age, male gender and lifestyle factors such as obesity and smoking. Inflammation has also been implicated in cellular senescence and may promote telomere shortening in chronic conditions such as obesity and diabetes. However, little is known about the relationship between markers of obesity and inflammation, and leukocyte telomere length (LTL).

Methods: LTL was measured using quantitative polymerase chain reaction in peripheral leukocytes from 295 individuals diagnosed with Barrett's esophagus (BE) between 1995 and 2009. Data on lifestyle variables including obesity and smoking were collected at in-person interviews. Biomarkers of obesity (leptin, adiponectin), diabetes (glucose, insulin), inflammation (C-reactive protein, Interleukin-6, surface tumor necrosis factor receptor (sTNFR) I & II) and oxidative stress (F2-isoprostanes) were measured in stored blood samples. We examined associations between these covariates and LTL in a cross-sectional analysis using linear and logistic regression models, adjusting for possible confounders.

Results: LTL was significantly associated with age (r = -0.30, p < 0.001), gender (r = 0.14 for females, p = 0.01) and inversely associated with cigarette pack-years (r = -0.11, p = 0.04). Odds of having short LTL were significantly higher for participants in the highest tertile for sTNF-RI (Odds ratio adjusted for age, gender, smoking, and obesity = 2.19; 95 % CI 1.00-4.85, p-trend = 0.02). LTL was not significantly associated with any other lifestyle factors, including smoking or obesity, or other inflammation-, obesity-/diabetes-related biomarkers measured.

Conclusions: Increasing age, male gender, smoking history, and sTNF-RI levels were associated with short LTL among persons with BE but no correlations were observed between LTL and other inflammatory markers or measures of obesity. Larger longitudinal studies are necessary in order to further establish the potential relationships between obesity, inflammation markers and LTL.
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http://dx.doi.org/10.1186/s40608-015-0063-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566310PMC
September 2015

Shorter Ends, Faster End? Leukocyte Telomere Length and Mortality Among Older Taiwanese.

J Gerontol A Biol Sci Med Sci 2015 Dec 17;70(12):1490-8. Epub 2014 Oct 17.

Department of Pathology, University of Washington, Seattle, Washington.

Recent studies have found mixed results regarding the association between leukocyte telomere length (LTL)--thought to be a marker of cellular aging--and all-cause mortality. Some studies have reported a significant inverse relationship, but others have not, perhaps in part owing to insufficient power. We examine the relationship using data from a nationally representative sample of older Taiwanese (54+ in 2000), which is larger (n = 942) than most previous studies, and which includes comprehensive information on potential confounders including white blood cell distribution and inflammatory markers. Results from a Cox hazards model demonstrate a small, but significant, association between LTL and mortality that is independent of age, sex, and lifestyle factors. White blood cell distribution, especially the proportion of neutrophils, is an important predictor of LTL; however, the association between LTL and mortality changes little controlling for white blood cell distribution. In contrast, the association between LTL and mortality weakens considerably (by 48%) after adjustment for inflammatory markers and homocysteine. Our results suggest that the relationship between short telomeres and mortality is tied to inflammation and homocysteine. Longitudinal studies are needed to explore bidirectional influences resulting from the fact that inflammation leads to shorter leukocyte telomeres, which in turn results in senescence, which exacerbates inflammation.
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http://dx.doi.org/10.1093/gerona/glu191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751225PMC
December 2015

Detecting ultralow-frequency mutations by Duplex Sequencing.

Nat Protoc 2014 Nov 9;9(11):2586-606. Epub 2014 Oct 9.

1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.

Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.
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http://dx.doi.org/10.1038/nprot.2014.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271547PMC
November 2014

Response.

J Natl Cancer Inst 2014 Feb 8;106(2):djt437. Epub 2014 Jan 8.

Affiliations of authors: Department of Pathology (RAR, CU, JJS, PSR), Department of Medicine (JJS, TAB), and Division of Gastroenterology (TAB), University of Washington, Seattle, WA; Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA (PSR).

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http://dx.doi.org/10.1093/jnci/djt437DOI Listing
February 2014

Clonal expansions and short telomeres are associated with neoplasia in early-onset, but not late-onset, ulcerative colitis.

Inflamm Bowel Dis 2013 Nov;19(12):2593-602

*Department of Pathology, †Department of Medicine, ‡Department of Biostatistics, and §Division of Gastroenterology, University of Washington, School of Medicine, Seattle, Washington; ‖Division of Anatomic Pathology, University of Utah, Salt Lake City, Utah; and ¶Division of Public Health Science, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background: Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case-control study.

Methods: This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics.

Results: Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity.

Conclusions: Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields.
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http://dx.doi.org/10.1097/MIB.0b013e3182a87640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3885330PMC
November 2013

Mitochondria and tumor progression in ulcerative colitis.

J Natl Cancer Inst 2013 Aug 12;105(16):1239-48. Epub 2013 Jul 12.

Department of Pathology, University of Washington, Seattle, WA, USA.

Background: The role of mitochondria in cancer is poorly understood. Ulcerative colitis (UC) is an inflammatory bowel disease that predisposes to colorectal cancer and is an excellent model to study tumor progression. Our goal was to characterize mitochondrial alterations in UC tumorigenesis.

Methods: Nondysplastic colon biopsies from UC patients with high-grade dysplasia or cancer (progressors; n = 9) and UC patients dysplasia free (nonprogressors; n = 9) were immunostained for cytochrome C oxidase (COX), a component of the electron transport chain, and were quantified by multispectral imaging. For six additional progressors, nondysplastic and dysplastic biopsies were stained for COX and additional mitochondrial proteins including PGC1α, the master regulator of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number was determined by quantitative polymerase chain reaction. Generalized estimating equations with two-sided tests were used to account for correlation of measurements within individuals.

Results: Nondysplastic biopsies of UC progressors showed statistically significant COX loss compared with UC nonprogressors by generalized estimating equation (-18.5 units, 95% confidence interval = -12.1 to -24.9; P < .001). COX intensity progressively decreased with proximity to dysplasia and was the lowest in adjacent to dysplasia and dysplastic epithelium. Surprisingly, COX intensity was statistically significantly increased in cancers. This bimodal pattern was observed for other mitochondrial proteins, including PGC1α, and was confirmed by mtDNA copy number.

Conclusions: Mitochondrial loss precedes the development of dysplasia, and it could be used to detect and potentially predict cancer. Cancer cells restore mitochondria, suggesting that mitochondria are needed for further proliferation. This bimodal pattern might be driven by transcriptional regulation of mitochondrial biogenesis by PGC1α.
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http://dx.doi.org/10.1093/jnci/djt167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748006PMC
August 2013

Independent and combined effects of dietary weight loss and exercise on leukocyte telomere length in postmenopausal women.

Obesity (Silver Spring) 2013 Dec 29;21(12):E549-54. Epub 2013 Jul 29.

Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Objective: Investigate the effects of 12 months of dietary weight loss and/or aerobic exercise on leukocyte telomere length in postmenopausal women.

Design And Methods: Four hundred and thirty nine overweight or obese women (50-75 years) were randomized to: (i) dietary weight loss (N = 118); (ii) aerobic exercise (N = 117), (iii) diet + exercise (N = 117), or (iv) control (N = 87). The diet intervention was a group-based program with a 10% weight loss goal. The exercise intervention was 45 min day(-1) , 5 days week(-1) of moderate-to-vigorous aerobic activity. Fasting blood samples were taken at baseline and 12 months. DNA was extracted from isolated leukocytes and telomere length was measured by quantitative-polymerase chain reaction (qPCR). Mean changes were compared between groups (intent-to-treat) using generalized estimating equations.

Results: Baseline telomere length was inversely associated with age (r = -0.12 P < 0.01) and positively associated with maximal oxygen uptake (r = 0.11, P = 0.03), but not with BMI or %body fat. Change in telomere length was inversely correlated with baseline telomere length (r = -0.47, P < 0.0001). No significant difference in leukocyte telomere length was detected in any intervention group compared to controls, nor was the magnitude of weight loss associated with telomere length at 12 months.

Conclusions: Twelve months of dietary weight loss and exercise did not change telomere length in postmenopausal women.
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http://dx.doi.org/10.1002/oby.20509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3786031PMC
December 2013

Pan-colonic field defects are detected by CGH in the colons of UC patients with dysplasia/cancer.

Cancer Lett 2012 Jul 2;320(2):180-8. Epub 2012 Mar 2.

Department of Medicine, University of Washington, Seattle, WA, United States.

BAC arrays were used to evaluate genomic instability along the colon of patients with ulcerative colitis (UC). Genomic instability increases with disease progression and biopsies more proximal to dysplasia showed increased instability. Pan-colonic field copy number gain or loss involving small (<1Mb) regions were detected in most patients and were particularly apparent in the UC progressor patients who had dysplasia or cancer. Chromosomal copy gains or losses affecting large regions were mainly restricted to dysplastic biopsies. Areas of significant chromosomal losses were detected in the UC progressors on chromosomes 2q36, 3q25, 3p21, 4q34, 4p16.2, 15q22, and 16p13 (p-value⩽0.04). These results extend our understanding of the dynamic nature of pan-colonic genomic instability in this disease.
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http://dx.doi.org/10.1016/j.canlet.2012.02.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406733PMC
July 2012

Ulcerative colitis-associated colorectal cancer arises in a field of short telomeres, senescence, and inflammation.

Cancer Res 2011 Mar;71(5):1669-79

Department of Pathology, University of Washington, Seattle, Washington 98195-7705, USA.

Inflammation plays a role in the progression to cancer and it is linked to the presence of senescent cells. Ulcerative colitis (UC) is a chronic inflammatory disease that predisposes to colorectal cancer. Tumorigenesis in this setting is associated with telomere shortening that can be observed in the nondysplastic epithelium of UC patients with high-grade dysplasia (HGD) or cancer (UC progressors). We hypothesized that a preneoplastic field of inflammation, telomere shortening, and senescence underlies tumor progression in UC progressors. Multiple biopsies of varying histologic grade were collected along the colon of nine UC progressors and analyzed for telomere length, DNA damage, senescence, p53, p16, and chronic and acute inflammation. Twenty biopsies from four UC nonprogressors and twenty-one biopsies from control individuals without UC were also analyzed. Short telomeres and increased DNA damage, senescence, and infiltrating leukocytes were observed in biopsies located less than 10 cm from HGD or cancer. Low-grade dysplasia (LGD) had the shortest telomeres along with the highest levels of senescence and infiltrating leukocytes, whereas HGD biopsies showed the opposite pattern. The expression of p16 and p53 was low in nondysplastic biopsies but progressively increased in LGD and HGD. In addition, high levels of infiltrating leukocytes were associated with telomere shortening, senescence, and reduced p53 expression. These results suggest that dysplasia arises in a preneoplastic field of chronic inflammation, which leads to telomere shortening, DNA damage, and senescence. Our findings argue that senescence acts as a tumor suppressor mechanism that is abrogated during the transition from LGD to HGD in UC.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-1966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077943PMC
March 2011

Leukocyte telomere length is associated with disability in older u.s. Population.

J Am Geriatr Soc 2010 Jul 23;58(7):1289-98. Epub 2010 Jun 23.

Department of Pathology, University of Washington, Seattle, WA 98195, USA.

Objectives: To determine whether mean leukocyte telomere length (LTL) serves as a biomarker of disability assessed according to activities of daily living (ADLs) and what factors may modify this relationship.

Design: Retrospective cross-sectional study.

Setting: A subset of the National Long Term Care Survey (NTLCS), a Medicare-based U.S. population longitudinal study focused on trends of overall health and functional status in older adults.

Participants: Six hundred and twenty-four individuals from the 1999 wave of the NTLCS cohort.

Measurements: Relative LTL determined according to quantitative polymerase chain reaction. LTL has previously been shown to correlate with common age-related disorders and mortality, as well as with socioeconomic status.

Results: A sex difference in LTL was observed but not age-dependent shortening or association with socioeconomic status. LTL was associated with disability and functional status assessed according to ADLs. The association between ADLs and LTL was stronger in subjects without diabetes mellitus, whereas associations were not seen when only subjects with diabetes mellitus were analyzed. Associations between LTL and cardiovascular disease (CVD) and cancer were also present in the group without diabetes mellitus but not in the group with diabetes mellitus.

Conclusion: These findings support the concept that LTL is a biomarker of overall well-being that is predictive of disability of older individuals in the U.S. population. Diabetes mellitus plays an important role as a modifier of the association between LTL and disability, CVD, and cancer. These associations have clinical implications because of the potential predictive value of LTL and deserve further investigation.
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http://dx.doi.org/10.1111/j.1532-5415.2010.02948.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2918372PMC
July 2010

Telomere length, pre-eclampsia, and gestational diabetes.

BMC Res Notes 2010 Apr 23;3:113. Epub 2010 Apr 23.

Department of Epidemiology, Tulane School of Public Health and Tropical Medicine, New Orleans, LA, USA.

Background: Telomere length is a marker of cumulative damage to the cell, and has been associated with cardiovascular disease, hypertension, and diabetes.

Findings: The association of telomere length with pre-eclampsia and gestational diabetes mellitus (GDM) was examined in a nested case-control study. Circulating leukocyte telomere length was measured by Quantitative-PCR. Mean and median telomere length among cases and controls was compared, and logistic regression was used to model the outcomes as a function of tertile telomere length, with control for effects of potential confounders. Mean telomere length in pre-eclampsia cases was 0.77 (SD 0.14), in GDM cases was 0.73 (SD 0.10), and in controls was 0.74 (SD 0.14). The adjusted odds ratio comparing the highest tertile to the lowest for pre-eclampsia was 0.92 (0.15-5.46), and for gestational diabetes was 0.65 (0.13-3.34).

Conclusions: Further study is necessary to determine if telomere length is associated with these pregnancy complications.
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http://dx.doi.org/10.1186/1756-0500-3-113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873349PMC
April 2010

Clonal expansions in ulcerative colitis identify patients with neoplasia.

Proc Natl Acad Sci U S A 2009 Dec 19;106(49):20871-6. Epub 2009 Nov 19.

Department of Pathology, University of Washington School of Medicine, Seattle, WA 98105, USA.

Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.
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http://dx.doi.org/10.1073/pnas.0909428106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779829PMC
December 2009

Quantitative fluorescence in situ hybridization (QFISH) of telomere lengths in tissue and cells.

Curr Protoc Cytom 2005 Aug;Chapter 12:Unit 12.6

St Vincent's University Hospital, Elm Park, Dublin, Ireland.

Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end-to-end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ-based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence-conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin-fixed paraffin-embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar-embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image-analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.
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http://dx.doi.org/10.1002/0471142956.cy1206s33DOI Listing
August 2005
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