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

Symptomatic women referred to a surgical oncologist for a clinical breast examination and who had a negative result were recruited for this study. A total of 20 women were selected for the cross validation study. All patients were recruited in accordance with the ethical guidelines of the Thunder Bay Regional Health Sciences Ethics Board in adherence to the Tri-Council Policy Statement on Ethical Conduct for Research Involving Humans. Written consent was obtained from the patients for publication of the study.

Blood samples were collected by standard venipuncture using a BD Vacutainer CPT™. Additionally, blood from a finger prick was collected on isocode cards from each patient. MtDNA was extracted from whole blood using a QiaAmp DNA MiniKit (Qiagen). NAF was recovered by a qualified practitioner, using a FIRSTCYTE™ Aspirator (Cytyc Health Corporation) following the recommendations of the manufacturer, and stored in CytoLyt Solution until extracted. Using this device, NAF was expressed from 1–3 ducts and pooled for the study. This method of NAF collection is therefore not representative of the entire ductal system, and could miss ducts with lesions. For diagnostic purposes, a better method of NAF collection is needed. The total volume of NAF collected per patient ranged from 50 – 100 μL. MtDNA was extracted using the QiaAmp DNA MiniKit (Qiagen) and archived at -86°C. Both NAF and blood isolated mtDNA samples were randomized and blinded for the cross-lab validation.

PCR clean up was conducted using the QIAquick 96 well vacuum plate manifold and protocol (Qiagen). DNAs were eluted into 65 uL of DNAse/RNAse free water.

Amplified mtgenome template was sent to CoGenics (Houston, Texas) for capillary electrophoresis sequencing. Briefly, Big Dye Termination Chemistry and a series of 72 primers were used to sequence in both forward and reverse directions. The fluorescent PCR-based sequencing was also conducted independently as a cross validation at NIST using previously published reaction conditions [2]. Primers contained M13 tags to facilitate DNA sequencing with M13 forward and reverse sequences. Briefly, the blood and NAF mtDNAs were sequenced using the Big Dye™ Terminator (BDT) version 3.1 cycle sequencing kit (ABI). A one eighth cycle sequencing reaction was used for each primer. Reactions contained 1 μL of each of the following reagents: BDT reagent, DNA (3–6 ng/μL), M13 primer (forward or reverse; 5 pmol/μL), 5× Dilution Buffer (ABI), and dH₂O to a final volume of 5 μL. Cycling sequencing conditions for forward primers were as follows: (40 cycles): 96°C for 10 s; annealing, 50°C for 5 s; elongation, 60°C for 4 min; 4°C hold. Reverse primers were sequenced using the same protocol, but the annealing temperature was lowered to 37°C.

The Montage™ SEQ₉₆ plate (Millipore Corp., Billerica, MA) was used for clean-up following the cycle sequencing reactions. Thirty microliters of Wash Solution was added to each well of the cycle sequencing plate. The samples were transferred to the clean-up plate and placed on the vacuum manifold for 15–20 minutes or until the wells were dry. A second wash of 30 μL Wash Solution was added and vacuumed dry for an additional 25 to 30 minutes. Once dry, 20 μL of Injection Solution were added to each well and the plate was mixed vigorously on a plate shaker for 10 minutes. Resuspended samples were transferred to a 3100 Optical Plate and diluted with 15 μL of HI-DI Formamide (ABI). All separations were performed using the ABI 3130×LGenetic Analyzer with an 80 cm capillary and POP7 polymer system. Samples were electrokinetically injected (30 seconds, 1 KV) and separated at 14.6 KV. Sequences were aligned using the DNA Star SeqMan II (5.05) program and scanned for polymorphisms and sequence variants in direct comparison to revised Cambridge Reference Sequence (rCRS) and the corresponding sequence of the blood.

The GeneChip^® CustomSeq^® Resquencing Array Protocol Version 2 was used with a few modifications. Following amplification of the mtgenome, template was prepared and hybridized as recommended by the GeneChip^® Resequencing Reagent Kit. MCv2 chips were processed in the GeneChip^® Hybridization Oven 640, GeneChip^® Fluidics Station 450 and the GeneChip^® Scanner 3000. Briefly, either three or nine amplicons representing the patient and normal control mitochondrial genomes were separately pooled at equi-molar concentrations. The PCR amplification products were pooled, fragmented, labeled, hybridized, washed, and scanned. The total quantity of DNA applied to the array was 0.62 μg. Fragmentation of the pooled DNAs was conducted using 0.15 units of Fragmentation reagent (0.033 μL) per sample at 37°Cfor 15 minutes followed by 95°C for 15 minutes to inactivate. The fragments were labeled with 30 units of TdT at 37°C for 90 minutes followed by 95°C for 15 minutes. The hybridization cocktail, including separately prepared control fragments, was hybridized for 16 to 18 hours at 45°C with 60 rpm. Arrays were washed using the mini_mapping10kv1_450 fluidics protocol, scanned on a GeneArray^® 2500 Scanner or a GeneChip^® Scanner 3000G7 Scanner, and analyzed with GeneChip^® DNA analysis (GDAS) and GSEQ Softwares.

Final analysis of all data was conducted using Affymetrix software GCOS v1.4 and GSEQ v4.0. The probe intensities for each mutation reported by the software were examined on the forward and reverse strands for every occurrence of a specific base position located on the chip. Mutations were confirmed and only reported when the mutation was seen on both strands for locations appearing once, and 3 of 4 strands for locations that appear on the chip twice. We selected a random subset of our patient samples (3 tissues each from 4 patients) to evaluate the accuracy of the MitoChip in comparison to CE DNA sequencing. All samples were blinded and phylogenetic trees were returned using the PHYLIP program Dnaml, Dnapars and Dnadist [27].

All samples were genotyped using the PowerPlex^® 16 System (Promega Corp, Madison, WI) on a 3130×L genetic analyzer with a 36 cm capillary array and POP4 polymer and analyzed using GeneMapper^® ID v3.2 (Applied Biosystems, Foster City, CA (ABI). Samples were diluted to 0.5 – 1.0 ng/μL and 1 μL of sample was added to a 24 μL reaction volume (18.2 μL H₂O, 2.5 μL 10× buffer, 2.5 μL PowerPlex^® 16 10× primer pair mix, 0.8 μL (4U) AmpliTaq Gold^® DNA Polymerase (ABI), then PCR amplified using published conditions. 1 μL of ILS600 internal lane standard and 9 μL of HiDi™ Formamide (ABI) were added to 1 μL of reaction (or 1 μL Allelic Ladder Mix, one for each run) then the mix was briefly denatured and chilled to 95°C and then placed on crushed ice for 3 minutes prior to each sequencing run.

STR typing was performed on all samples to eliminate the possibility of inadvertent sample mix-up or cross contamination.

Two previous studies have examined mtDNA mutations in NAF, however, both studies examined less than 30% of the mtgenome [18, 19]. For diagnostic purposes, it is more informative to interrogate the complete mtgenome. Hence, our attempt was to reliably sequence the complete mtgenome from NAF. NAF was successfully extracted from 23 of the 28 women recruited for this study. Twenty two (96%) of the women produced NAF from both breasts, and 20 NAF samples from the left breasts and matched blood were chosen for this study. Table 2 shows the clinicopathologic and demographic data of the study participants. Overall thirty-nine (20 blood and 19 NAF) samples were successfully processed; one NAF sample could not be fully amplified and was not included in the analysis. The entire mtgenome of 14 out of 19 NAF samples were successfully amplified using a single full length primer pair, with the remaining 5 samples requiring three overlapping primers. Although the primers were designed to preclude pseudogene co-amplification, the ability to amplify the full length mtgenome in biological samples such as NAF with reduced cellularity is an added quality assurance measure against pseudogenes. Sequence comparison between the NAF, blood and rCRS revealed a total of 490 polymorphisms and 7 mutations. Of the 7 mutations (SNPs discovered only in the NAF and not present in the blood) noted between all samples, three were associated with known problematic features of MCv2 at nucleotide positions 9179, 9914, and 11719 (Table 3). Comparison of MCv2 sequence data to the gold standard (CE) revealed a 99.999% identity. These results indicate that microarray resequencing of the mtgenome recovered from NAF is a rapid, sensitive and cost effective technique. Importantly, this technology enables clinical access to the information content of the mtgenome of mammary ductal epithelial cells.

The ability to rapidly sequence the mtgenome from NAF was cross-lab validated by independent confirmation at separate venues with a 99.200% concordance. Table 3 records the comparative data between the work done at NIST and Genesis Genomics, demonstrating repeatability of the detection method and consensus on the number of mtDNA mutations. Maximum likelihood clustering analysis of the blinded results from both labs demonstrates general clustering from the suite of samples from any given subject (Figure 1), again demonstrating the accuracy of the data obtained at each institution. Moreover, each individual was resolved into mitochondrial haplogroups, as expected. Haplogroup polymorphisms were accurately detected in both NAF and blood samples from the respective participant. In general, this study population was essentially of European ancestry. Although Figure 1 appears to indicate differences between the NAF and blood samples from a given patient, this effect is an artifact since the genetic distances between these samples is very small (e.g. 0.00001). These small distances are also true for the general outline of the tree.

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Four out of 19 (21%) NAF samples contained a single point mutation difference from the matched controls (blood) (Table 4). Previous work on partial mtgenome analysis is consistent with our results [18]. This work by Zhu et al. reported that some mutations detected in NAF were also found in the patient's primary tumor, emphasizing the possible utility of NAF for breast cancer screening. Although no tumor was detected in our cohort of women, these mutations may indicate mtgenome instability, perhaps suggesting an elevated risk for breast cancer in these 4 women. For example, patient 1069 had a C/T heteroplasmy at bp 516, which is within a D-Loop region of known mtgenome instability in breast tumors [28]. Heteroplasmy is considered an early marker of potential disease [29, 30]. Moreover, one study concluded that breast tumors that have mutations within the D-Loop have less favorable patient outcomes than those lacking these alterations [31]. Thus this simple, sensitive measurement of the mutation load from non-invasively collected samples, offers an alternative for diagnostic purposes.

One important aspect of this project is the massive amount of genetic data collected in a relatively short period. Close to 1.6 × 10⁶ mtgenome bases were sequenced, demonstrating an extremely fine genetic resolution when using the MCv2. This suggests that cross-validated point mutations may indeed be important indicators of altered molecular processes indicative of potential transformation. Independent cross-validation confirmed these mutations. Combined with the broad resolution demonstrated by the blinded Maximum likelihood clustering results, in association with corresponding demographic data, this technology is capable of gathering a broad spectrum of population level data. Moreover, this methodology may have important utility in the early detection of breast cancer associated mutations in NAF.