Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0
| Autor: | Kenneth E. Gehman, Paul D. Wagner, Teresa Gehman, Robert Thayer, Sudhir Srivastava, Kerry Robinson, Roy Wittock, Samantha Maragh, Alioune Ngom, Andrea Maggrah, Jennifer Maki, Ryan Parr, Katrina Maki, Gabriel D. Dakubo, John P. Jakupciak, Brian Reguly |
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| Rok vydání: | 2008 |
| Předmět: |
Adult
Cancer Research Pathology medicine.medical_specialty Mitochondrial DNA Mutation rate Somatic cell Sequence analysis DNA Mutational Analysis Biology medicine.disease_cause DNA Mitochondrial lcsh:RC254-282 Germline Breast Diseases Breast cancer Genetics medicine Humans Oligonucleotide Array Sequence Analysis Mutation Biopsy Needle Middle Aged lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens medicine.disease Molecular biology Body Fluids Mitochondria body regions Oncology Nipples Genome Mitochondrial Feasibility Studies Female Primer (molecular biology) Research Article |
| Zdroj: | BMC Cancer, Vol 8, Iss 1, p 95 (2008) BMC Cancer |
| ISSN: | 1471-2407 |
| DOI: | 10.1186/1471-2407-8-95 |
| Popis: | Background Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Nipple aspirate fluid (NAF) from symptomatic women could potentially serve as a minimally invasive sample for breast cancer screening by detecting somatic mutations in this biofluid. This study is aimed at 1) demonstrating the feasibility of NAF recovery from symptomatic women, 2) examining the feasibility of sequencing the entire mitochondrial genome from NAF samples, 3) cross validation of the Human mitochondrial resequencing array 2.0 (MCv2), and 4) assessing the somatic mtDNA mutation rate in benign breast diseases as a potential tool for monitoring early somatic mutations associated with breast cancer. Methods NAF and blood were obtained from women with symptomatic benign breast conditions, and we successfully assessed the mutation load in the entire mitochondrial genome of 19 of these women. DNA extracts from NAF were sequenced using the mitochondrial resequencing array MCv2 and by capillary electrophoresis (CE) methods as a quality comparison. Sequencing was performed independently at two institutions and the results compared. The germline mtDNA sequence determined using DNA isolated from the patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's NAF. Results From the cohort of 28 women recruited for this study, NAF was successfully recovered from 23 participants (82%). Twenty two (96%) of the women produced fluids from both breasts. Twenty NAF samples and corresponding blood were chosen for this study. Except for one NAF sample, the whole mtgenome was successfully amplified using a single primer pair, or three pairs of overlapping primers. Comparison of MCv2 data from the two institutions demonstrates 99.200% concordance. Moreover, MCv2 data was 99.999% identical to CE sequencing, indicating that MCv2 is a reliable method to rapidly sequence the entire mtgenome. Four NAF samples contained somatic mutations. Conclusion We have demonstrated that NAF is a suitable material for mtDNA sequence analysis using the rapid and reliable MCv2. Somatic mtDNA mutations present in NAF of women with benign breast diseases could potentially be used as risk factors for progression to breast cancer, but this will require a much larger study with clinical follow up. |
| Databáze: | OpenAIRE |
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