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Andrea Maggrah, Kerry Robinson,
Jennifer Creed,
Roy Wittock,
Ken Gehman,
Teresa Gehman,
Helen Brown,
Andrew Harbottle,
M Kent Froberg,
Daniel Klein,
Brian Reguly,
Ryan Parr
[show abstract]
[hide abstract]
ABSTRACT: Alterations in the mitochondrial genome have been chronicled in most solid tumors, including breast cancer. The intent of this paper is to compare and document somatic mitochondrial D-loop mutations in paired samples of ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) indicating a potential breast ductal epithelial cancerization field effect. Paired samples of these histopathologies were laser-captured microdissected (LCM) from biopsy, lumpectomy, and mastectomy tissues. Blood samples were collected as germplasm control references. For each patient, hypervariable region 1 (HV1) in the D-loop portion of the mitochondrial genome (mtGenome) was sequenced for all 3 clinical samples. Specific parallel somatic heteroplasmic alterations between these histopathologies, particularly at sites 16189, 16223, 16224, 16270, and 16291, suggest the presence of an epithelial, mitochondrial cancerization field effect. These results indicate that further characterization of the mutational pathway of DCIS and IBC may help establish the invasive potential of DCIS. Moreover, this paper indicates that biofluids with low cellularity, such as nipple aspirate fluid and/or ductal lavage, warrant further investigation as early and minimally invasive detection mediums of a cancerization field effect within breast tissue.
BioMed research international. 01/2013; 2013:379438.
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John P Jakupciak,
Andrea Maggrah,
Samantha Maragh,
Jennifer Maki,
Brian Reguly,
Katrina Maki,
Roy Wittock, Kerry Robinson,
Paul D Wagner,
Robert E Thayer,
Ken Gehman,
Teresa Gehman,
Sudhir Srivastava,
Alioune Ngom,
Gabriel D Dakubo,
Ryan L Parr
[show abstract]
[hide abstract]
ABSTRACT: 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.
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.
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.
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.
BMC Cancer 02/2008; 8:95. · 3.01 Impact Factor
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Jennifer Maki, Kerry Robinson,
Brian Reguly,
Jude Alexander,
Roy Wittock,
Andrea Aguirre,
Eleftherios P Diamandis,
Nicholas Escott,
Anthony Skehan,
Owen Prowse,
Robert E Thayer,
M Kent Froberg,
Michael J Wilson,
Samantha Maragh,
John P Jakupciak,
Paul D Wagner,
Sudhir Srivastava,
Gabriel D Dakubo,
Ryan L Parr
[show abstract]
[hide abstract]
ABSTRACT: We report the usefulness of a 3.4-kb mitochondrial genome deletion (3.4 mtdelta) for molecular definition of benign, malignant, and proximal to malignant (PTM) prostate needle biopsy specimens. The 3.4 mtdelta was identified through long-extension polymerase chain reaction (PCR) analysis of frozen prostate cancer samples. A quantitative PCR assay was developed to measure the levels of the 3.4 mtdelta in clinical samples. For normalization, amplifications of a nuclear target and total mitochondrial DNA were included. Cycle threshold data from these targets were used to calculate a score for each biopsy sample. In a pilot study of 38 benign, 29 malignant, and 41 PTM biopsy specimens, the difference between benign and malignant core biopsy specimens was well differentiated (P & .0001), with PTM indistinguishable from malignant samples (P = .833). Results of a larger study were identical. In comparison with histopathologic examination for benign and malignant samples, the sensitivity and specificity were 80% and 71%, respectively, and the area under a receiver operating characteristic (ROC) curve was 0.83 for the deletion. In a blinded external validation study, the sensitivity and specificity were 83% and 79%, and the area under the ROC curve was 0.87. The 3.4 mtdelta may be useful in defining malignant, benign, and PTM prostate tissues.
American Journal of Clinical Pathology 01/2008; 129(1):57-66. · 2.60 Impact Factor
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John Jakupciak,
Andrea Maggrah,
Samantha Maragh,
Jennifer Maki,
Brian Reguly,
Katrina Maki,
Roy Wittock, Kerry Robinson,
Paul Wagner,
Robert Thayer,
Ken Gehman,
Teresa Gehman,
Sudhir Srivastava,
Alioune Ngom,
Gabriel Dakubo,
Ryan Parr
[show abstract]
[hide abstract]
ABSTRACT: Abstract
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.
BMC Cancer. 01/2008;
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Ryan L Parr,
Gabriel D Dakubo,
Keith A Crandall,
Jennifer Maki,
Brian Reguly,
Andrea Aguirre,
Roy Wittock, Kerry Robinson,
Jude S Alexander,
Mark A Birch-Machin,
Mamdouh Abdel-Malak,
M Kent Froberg,
Eleftherios P Diamandis,
Robert E Thayer
[show abstract]
[hide abstract]
ABSTRACT: Studies of somatic mitochondrial DNA mutations have become an important aspect of cancer research because these mutations might have functional significance and/or serve as a biosensor for tumor detection. Here we report somatic mitochondrial DNA mutations from three specific tissue types (tumor, adjacent benign, and distant benign) recovered from 24 prostatectomy samples. Needle biopsy tissue from 12 individuals referred for prostate biopsy, yet histologically benign (symptomatic benign), were used as among individual control samples. We also sampled blood (germplasm tissue) from each patient to serve as within individual controls relative to the somatic tissues sampled (malignant, adjacent, and distant benign). Complete mitochondrial genome sequencing was attempted on each sample. In contrast to both control groups [within patient (blood) and among patient (symptomatic benign)], all of the tissue types recovered from the malignant group harbored significantly different mitochondrial DNA (mtDNA) mutations. We conclude that mitochondrial genome mutations are an early indicator of malignant transformation in prostate tissue. These mutations occur well before changes in tissue histo-pathology, indicative of prostate cancer, are evident to the pathologist.
Journal of Molecular Diagnostics 08/2006; 8(3):312-9. · 3.58 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.
Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero (rho0) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences.
These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.
BMC Genomics 02/2006; 7:185. · 4.07 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Abstract
Background
Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.
Results
Here we report the cloning and sequencing of numts loci, amplified from human tissue and rho-zero ( ρ <sup>0</sup>) cells (control) with primers known to amplify the mitochondrial genome. This paper is the first to fully sequence 46 paralogous nuclear DNA fragments that represent the entire mitochondrial genome. This is a surprisingly small number due primarily to the primer sets used in this study, because prior to this, BLAST searches have suggested that nuclear DNA harbors between 400 to 1,500 paralogous mitochondrial DNA fragments. Our results indicate that multiple numts were amplified simultaneously with the mitochondrial genome and increased the load of pseudogene signal in PCR reactions. Further, the entire mitochondrial genome was represented by multiple copies of paralogous nuclear sequences.
Conclusion
These findings suggest that mitochondrial genome disease-associated biomarkers must be rigorously authenticated to preclude any affiliation with paralogous nuclear pseudogenes. Importantly, the common perception that mitochondrial template "swamps" numts loci precluding detectable amplification, depends on the region of the mitochondrial genome targeted by the PCR reaction and the number of pseudogene loci that may co-amplify. Cloning and relevant sequencing data will facilitate the correct interpretation. This is the first complete, wet-lab characterization of numts that represent the entire mitochondrial genome.
BMC Genomics. 01/2006;
