Samantha Maragh

Performance of mitochondrial DNA mutations detecting early stage cancer

BackgroundMutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites.MethodsWe determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region.ResultsUsing this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors.ConclusionOur findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is unclear the biological relevance of these detected mitochondrial mutations. Whether the detection of tumor-specific mtDNA mutations in body fluidsy this method will be useful for diagnosis and monitoring applications requires further investigation.

Mitochondrial Genome Deletion Aids in the Identification of False- and True-Negative Prostate Needle Core Biopsy Specimens

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.

Facile whole mitochondrial genome resequencing from nipple aspirate fluid using MitoChip v2.0

BackgroundMutations 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.MethodsNAF 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 patients blood (control) was compared to the mutations present in cellular mtDNA recovered from patients NAF.ResultsFrom 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.ConclusionWe 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.

Identification of RNA binding motif proteins essential for cardiovascular development

BackgroundWe recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis.ResultsTo determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b), and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA), posterior caudal vein (PCV) and caudal vein (CV) which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf). Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf.ConclusionTaken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

Development of genomic reference materials for Huntington disease genetic testing

Purpose: Diagnostic and predictive testing for Huntington disease requires an accurate measurement of CAG repeats in the HD (IT15) gene. However, precise repeat sizing can be technically challenging, and is complicated by the lack of quality control and reference materials (RM). The aim of this study was to characterize genomic DNA from 14 Huntington cell lines available from the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Cell Repositories for use as reference materials for CAG repeat sizing.Methods: Fourteen Huntington cell lines were selected for study. The alleles in these materials represent a large range of sizes that include important diagnostic cutoffs and allele combinations. The allele measurement study was conducted by ten volunteer laboratories using a variety of polymerase chain reaction-based in-house developed methods and by DNA sequence analysis.Results: The Huntington alleles in the 14 genomic DNA samples range in size from 15 to 100 CAG repeats. There was good agreement among the ten laboratories, and thus, the 95% confidence interval was small for each measurement. The allele size determined by DNA sequence analysis agreed with the laboratory developed tests.Conclusion: These DNA materials, which are available from Coriell Cell Repositories, will facilitate accurate and reliable Huntington genetic testing.

Multiple strand displacement amplification of mitochondrial DNA from clinical samples

BackgroundWhole genome amplification (WGA) methods allow diagnostic laboratories to overcome the common problem of insufficient DNA in patient specimens. Further, body fluid samples useful for cancer early detection are often difficult to amplify with traditional PCR methods. In this first application of WGA on the entire human mitochondrial genome, we compared the accuracy of mitochondrial DNA (mtDNA) sequence analysis after WGA to that performed without genome amplification. We applied the method to a small group of cancer cases and controls and demonstrated that WGA is capable of increasing the yield of starting DNA material with identical genetic sequence.MethodsDNA was isolated from clinical samples and sent to NIST. Samples were amplified by PCR and those with no visible amplification were re-amplified using the Multiple Displacement Amplificaiton technique of whole genome amplification. All samples were analyzed by mitochip for mitochondrial DNA sequence to compare sequence concordance of the WGA samples with respect to native DNA. Real-Time PCR analysis was conducted to determine the level of WGA amplification for both nuclear and mtDNA.ResultsIn total, 19 samples were compared and the concordance rate between WGA and native mtDNA sequences was 99.995%. All of the cancer associated mutations in the native mtDNA were detected in the WGA amplified material and heteroplasmies in the native mtDNA were detected with high fidelity in the WGA material. In addition to the native mtDNA sequence present in the sample, 13 new heteroplasmies were detected in the WGA material.ConclusionGenetic screening of mtDNA amplified by WGA is applicable for the detection of cancer associated mutations. Our results show the feasibility of this method for: 1) increasing the amount of DNA available for analysis, 2) recovering the identical mtDNA sequence, 3) accurately detecting mtDNA point mutations associated with cancer.

Rbm24a and Rbm24b are required for normal somitogenesis.

We recently demonstrated that the gene encoding the RNA binding motif protein 24 (RBM24) is expressed during mouse cardiogenesis, and determined the developmental requirement for its zebrafish homologs Rbm24a and Rbm24b during cardiac development. We demonstrate here that both Rbm24a and Rbm24b are also required for normal somite and craniofacial development. Diminution of rbm24a or rbm24b gene products by morpholino knockdown resulted in significant disruption of somite formation. Detailed in situ hybridization-based analyses of a spectrum of somitogenesis-associated transcripts revealed reduced expression of the cyclic muscle pattering genes dlc and dld encoding Notch ligands, as well as their respective target genes her7, her1. By contrast expression of the Notch receptors notch1a and notch3 appears unchanged. Some RBM-family members have been implicated in pre-mRNA processing. Analysis of affected Notch-pathway mRNAs in rbm24a and rbm24b morpholino-injected embryos revealed aberrant transcript fragments of dlc and dld, but not her1 or her7, suggesting the reduction in transcription levels of Notch pathway components may result from aberrant processing of its ligands. These data imply a previously unknown requirement for Rbm24a and Rbm24b in somite and craniofacial development. Although we anticipate the influence of disrupting RBM24 homologs likely extends beyond the Notch pathway, our results suggest their perturbation may directly, or indirectly, compromise post-transcriptional processing, exemplified by imprecise processing of dlc and dld.

Evaluation of two mitochondrial DNA biomarkers for prostate cancer detection.

BACKGROUND A 3.4kb deletion (3.4kbΔ ) in mitochondrial DNA (mtDNA) found in histologically normal prostate biopsy specimens has been reported to be a biomarker for the increased probability of prostate cancer. Increased mtDNA copy number is also reported as associated with cancer. OBJECTIVE Independent evaluation of these two potential prostate cancer biomarkers using formalin-fixed paraffin-embedded (FFPE) prostate tissue and matched urine and serum from a high risk cohort of men with and without prostate cancer. METHODS Biomarker levels were detected via qPCR. RESULTS Both 3.4kbΔ and mtDNA levels were significantly higher in cancer patient FFPE cores (p= 0.045 and p= 0.070 respectively at > 90% confidence). Urine from cancer patients contained significantly higher levels of mtDNA (p= 0.006, 64.3% sensitivity, 86.7% specificity). Combining the 3.4kbΔ and mtDNA gave better performance of detecting prostate cancer than either biomarker alone (FFPE 73.7% sensitivity, 65% specificity; urine 64.3% sensitivity, 100% specificity). In serum, there was no difference for any of the biomarkers. CONCLUSIONS This is the first report on detecting the 3.4kbΔ in urine and evaluating mtDNA levels as a prostate cancer biomarker. A confirmation study with increased sample size and possibly with additional biomarkers would need to be conducted to corroborate and extend these observations.