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

John P Jakupciak,Kerry Robinson,Teresa Gehman,Ken Gehman,Ryan L Parr,Jennifer Maki,Gabriel D Dakubo,Robert E Thayer,Samantha Maragh,Andrea Maggrah,Sudhir Srivastava,Paul D Wagner,Brian Reguly,Alioune Ngom,Roy Wittock,Katrina Maki

doi:10.1186/1471-2407-8-95

Abstract

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 patient's blood (control) was compared to the mutations present in cellular mtDNA recovered from patient's 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.

Highlights

Mutations in the mitochondrial genome have been associated with many disorders, including breast cancer
We report the feasibility of sequencing the complete mtgenome from nipple aspirate fluid (NAF), and the reliability of microarray based resequencing of the mtgenome to capillary electrophoresis (CE) sequencing technology as well as the reproducibility of the resequencing methodology in a cross validation study between the National Institute of Standards and Technology (Gaithersburg, MD USA) and Genesis Genomics Inc. (Thunder Bay, On Canada)
Sequencing of whole mtgenome from NAF samples Two previous studies have examined mitochondrial DNA (mtDNA) mutations in NAF, both studies examined less than 30% of the mtgenome [18,19]

Summary

Introduction

Mutations in the mitochondrial genome (mtgenome) have been associated with many disorders, including breast cancer. Research methods for identifying the presence of cancerous cells by measuring mutations in mtDNA is the subject of intense clinical investigation [1,2]. These studies analyze only specific regions of mtDNA and not the entire mitochondrial genome (mtgenome). There are several biological characteristics of mitochondria, and in particular the mtgenome that make it suitable for early detection and monitoring of neoplasia This genome has an accelerated mutation rate in comparison to the nuclear genome and accrues somatic mutations in tumor tissues [2,3,4,5]. The mitochondrial genome has a high copy number in comparison to the nuclear archive of DNA; there are potentially 1,000s of mtgenomes per cell, which enables easy detection of important biomarkers, even when only low amounts of samples are available

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