Quantification of DNA in Neonatal Dried Blood Spots by Adenine

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Quantification of DNA in neonatal dried blood spots by adenine tandem mass spectrometry Danielle Durie, Ed Yeh, Nathan McIntosh, Lawrence Fisher, Dennis E Bulman, H. Chaim Birnboim, Pranesh Chakraborty, and Osama Y Al-Dirbashi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03265 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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Analytical Chemistry

Quantification of DNA in neonatal dried blood spots by adenine tandem mass spectrometry Danielle Durie1, Ed Yeh1, Nathan McIntosh1, Lawrence Fisher1, Dennis E. Bulman1,2,3,4, H. Chaim Birnboim5, Pranesh Chakraborty1,2,4 and Osama Y. Al-Dirbashi1,4,6,* 1

Newborn Screening Ontario, Ottawa, Ontario, K1H 8L1, Canada Department of Pediatrics, University of Ottawa, Ontario, K1H8M5, Canada 3 Ottawa Hospital Research Institute, Ottawa, Ontario, K1H 8L6, Canada 4 Research Institute, Children’s Hospital of Eastern Ontario, Ottawa, Ontario, K1H 5B2, Canada 5 No affiliation 6 College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, 17172, United Arab Emirates 2

Newborn screening programs have expanded to include molecular-based assays as first-tier tests and the success of these assays depends on the quality and yield of DNA extracted from neonatal dried blood spots (DBS). To meet high throughput and rapid turnaround time requirements, newborn screening laboratories adopted rapid DNA extraction methods that produce crude extracts. Quantification of DNA in neonatal DBS is not routinely performed due to technical challenges; however, this may enhance the performance of assays that are sensitive to amounts of input DNA. In this study, we developed a novel high throughput method to quantify total DNA in DBS. It is based on specific acid-catalyzed depurination of DNA followed by mass spectrometric quantification of adenine. The amount of adenine was used to calculate DNA quantity per 3.2 mm DBS. Reference intervals were established using archived, neonatal DBS (n=501) and a median of 130.6 ng of DNA per DBS was obtained, which is in agreement with literature values. The intra- and inter-day variations were 92.2 and m/z 141.2 > 95.2 for adenine and 15N5-adenine, respectively, with a dwell time of 0.250 sec. The total run time was 1.3 min. ATP analysis was performed by chromatographic separation using porous graphitic carbon column and detected by negative-ion mode ESI -MS/MS as previously described23. Sample preparation Single 3.2 mm disc was punched from each DBS specimen. These discs were washed 5 times with 100 µL LC-MS grade water for 5 min on a nutator with mixing speed of 24 rpm at room temperature. After adding 100 µL of 0.2 mol/L HCl containing 0.7 µmol/L 15N5-adenine, samples were incubated for 1 hr at 60°C and 700 rpm to liberate adenine from DNA22. The extracts were removed from the discs and evaporated to dryness under vacuum at 45°C. The residue was reconstituted in 100 µL of mobile phase for mass spectrometric analysis. For measurement of DNA from DBS extracts, a single 3.2 mm disc was punched and DNA was extracted according to our inhouse routine procedure for PCR-based assays. After drying the extracts under vacuum, adenine was released from DNA and prepared for mass spectrometric analysis according to the procedure described above. For ATP measurements, single 3.2 mm DBS circles were punched into a 96-well plate and washed 5 times with 100 µL LC-MS grade water for 5 min on a nutator with mixing speed of 24 rpm at room temperature. DBS was then incubated with 100 µL of 20% methanol containing 1.0 µmol/L 13C10-ATP for 15 min at 23°C and 700 rpm. The resultant supernatant was then analyzed as described23. Method validation

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Analytical Chemistry Quantification of adenine in DBS was performed using a sixpoint standard curve. A solution of adenine in mobile phase was prepared by serial dilution and added to vacuum-dried DBS extracts (100 µL) to achieve the following concentrations: 0.16, 0.31, 0.63, 1.25, 2.5 and 5.0 µmol/L. These samples and a non-enriched DBS extract were treated and analyzed as described above. The adenine to 15N5-adenine peak area ratio obtained from non-enriched DBS extracts were subtracted from each standard. The corrected peak area ratio was plotted against the added adenine concentration. Limit of detection (LOD) and limit of quantification (LOQ) were defined as the adenine concentration that gave signal to noise ratio (S/N) ≥ 3 and S/N ≥ 10, respectively. The adenine concentration obtained from the calibration curve was converted to nanograms of DNA using the equation described by De Bruin and Birnboim (2016)22. This equation assumes that input DNA is double stranded, the mass of DNA is given as the sodium salt and other sources of adenine are negligible22. A GC content of 41% for human DNA was used in our calculations24. Intra-day (n=20) and inter-day (n=10) variations were tested using donor DBS samples. Coefficient of variation (CV%) was calculated according to the following equation [CV%=100 x standard deviation/mean]. Quality control (QC) material (DBS extracts spiked with high (5.0 µmol/L) and low (0.31 µmol/L) concentrations of adenine) was used to determine the analytical recovery which was calculated as follows [Recovery % = 100 x (concentration measured – concentration in non-enriched sample)/concentration added]. Recovery of DNA from DBS after our routine extraction was also quantified (n=67). One single 3.2 mm disc was used to determine the amount of DNA from DBS, and a second disc from the same sample was used to determine the DNA amount after extraction. Recovery is expressed as percentage of DNA in the extract compared to the original DBS sample. Quantification of adenine and DNA in neonatal DBS samples To determine the reference interval of DNA in neonatal DBS samples, we tested archived Newborn Screening Ontario DBS samples (n=501). These were randomly selected, screennegative samples and were anonymized. Samples were extracted for adenine and analyzed by ESI-MS/MS alongside adenine standard curve samples and QC material. Statistical analysis Wilcoxon matched-pairs signed rank test (two-sided, 95% confidence interval) was used to determine statistical significance between paired data (GraphPad, Prism 5). RESULTS ESI-MS/MS experiments MS/MS conditions were established using standard solutions of adenine and 15N5-adenine. Individual solutions of these compounds were infused continuously into the first quadrupole of the MS/MS system. ESI-MS scanning in the positive-ion mode revealed intense ions at m/z 136.1 and 141.2 corresponding to the [MH]+ of adenine and 15N5-adenine, respectively. These ions were subsequently transmitted to the collision cell and product ion scanning in the second resolving quadrupole was performed. Adenine produced three intense fragments at m/z 119.1, 92.2 and 65.2. These ions correspond to the loss of NH3, NH3-HCN and NH3-2HCN, respectively25. Under optimum conditions, 136.1 > 92.2 was the most abundant transition. The corresponding 15N5-adenine transition was 141.2 > 95.2. These specific transitions were free of interference and were selected for subsequent experiments.

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Figure 1 shows the product ion spectra of adenine and 15N5adenine.

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Figure 1. ESI-MS/MS product ion spectra of (a) adenine (m/z 136) and (b) 15N5-adenine (m/z 141).

In this work, chromatographic separation of adenine was not necessary and samples were introduced into the MS/MS using flow injection analysis. A flow rate gradient that changes the flow rate of 70% (v/v) acetonitrile containing 0.1% formic acid between 10-500 µL/min over the course of the run to maximize the sensitivity was employed. The use of flow surge at the end of each run reduced ion suppression and enhanced the peak shape. The analytical time between successive injections was 1.3 min. Sample preparation Compared with blank matrix preparations, the signal intensity of 15N5-adenine was suppressed by more than 90% in the presence of DBS extract. A washing step prior to sample extraction was therefore introduced. Various aqueous and/or organic mixtures composed of methanol, ethanol, 0.2 mol/L HCl, Tris buffer (10 mmol/L, pH 9.0) and water were attempted (Table S-1). We found that washing the DBS five times with 100 µL portions of LC-MS grade water gave the best results. Mean 15N5-adenine signal intensities of 7,638 and 156,132 were obtained from unwashed and washed DBS extracts, respectively, representing a 20-fold signal improvement (Fig. 2a). The purpose of washing is twofold, the ion suppression caused by blood components is reduced and 99.9% of ATP is removed from the DBS, eliminating it as a source of adenine in our analysis (Fig. 2b).

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Figure 2. Washing DBS prior to extraction reduces ion suppression and removes ATP. (a) Effect of washing DBS on the signal intensity of 15N5-adenine (n=11). (b) Effect of washing DBS on the signal intensity of ATP (n=3). Data represent mean signal intensity +/- standard error of the mean. Wilcoxon matched-pairs signed rank test: *P < 0.05 and ***P < 0.001 (two-sided with 95% confidence interval; GraphPad Prism 5).

Table 1. Intra- and inter-day reproducibility of adenine in DBS Intra-day (n=20) a

Inter-day (n=10) b

Sample

Mean (µmol/L)

SD (µmol/L)

CV (%)

Mean (µmol/L)

SDa (µmol/L)

CVb (%)

DBS 1

0.621

0.034

5.54

0.542

0.081

14.90

DBS 2

0.490

0.027

5.46

0.426

0.063

14.83

a

SD = standard deviation

b

CV (%) = coefficient variation

Table 2. Reference intervals of adenine and DNA per DBS adenine

Sample

(µmol/L)

DNA / DBS (ng)

(ng)

[12][26]

Estimated range in newborns

79 - 673

Neonatal DBS (n= 501) Median th

th

5 - 95 percentile 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304

1.07

14.5

130.6

0.55 – 1.82

7.5 – 24.6

67.1 – 221.2

In subsequent experiments, washing the DBS five times, each wash 5 min with 100 µL LC-MS grade water, at room temperature and 24 rpm was performed prior to adenine extraction. Extraction of adenine from washed DBS was achieved by acid treatment with 100 µL 0.2 mol/L HCl containing 0.7 µmol/L 15N5-adenine and incubation for 1 hr at 60°C and 700 rpm22. Adenine was stable for at least 24 h when stored at 2-8 ºC. Assay validation Linearity was established using DBS extracts enriched with adenine at 0.16, 0.31, 0.63, 1.25, 2.50 and 5.00 µmol/L. Nonenriched DBS extract was included in every batch to correct for endogenous levels of adenine. Regression analysis over the studied range showed a linear relationship (y = 1.23x + 0.0011, r2=0.999) with y as area ratio of adenine to 15N5-adenine and x as the added adenine concentration (µmol/L). The LOD (S/N > 3) and LOQ (S/N > 10) were calculated to be 12.5 and 37.8 nmol/L adenine, respectively. Intra-day (n=20) and Inter-day (n=10) imprecisions of adenine measurements were evaluated by repeated analysis of DBS samples. Table 1 summarizes the imprecision expressed as coefficient of variation (%). The analytical recovery evaluated

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in triplicate analysis at 0.31 and 5.0 µmol/L adenine per DBS ranged between 93-104%. Using the quantity of DNA in DBS and in DBS extracts (n=67) determined by the current method, we calculated the recovery of DNA of our in-house routine DNA extraction method to be 102%. Reference intervals of adenine and DNA in DBS The median (5th-95th percentile interval) of adenine per DBS obtained by the current method using neonatal samples from healthy individuals (n=501) was 14.5 ng (7.5 - 24.6 ng). This corresponds to 130.6 ng (67.1 - 221.2 ng) of DNA per DBS (Table 2). DISCUSSION In the past decade, molecular methods have been adopted as first-tier screening tests in NBS laboratories5. In general, success of molecular screening assays depends on input DNA quantity, integrity and purity; result interpretation hinges on the assumption that these factors are adequate for samples tested. Currently, NBS laboratories do not quantify DNA extracted from DBS prior to downstream molecular testing. This might be attributed to the fact that current quantification methods are unreliable, require significant additional time,

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Analytical Chemistry require manual setup and are an additional cost. Some molecular assays successfully incorporate an indicator of DNA quality within the assay itself (e.g. β-actin in a T-cell receptor excision circle qPCR assay)27. However, this approach may not be universally possible for all molecular assays because of defined limits of targets based on available dye sets and the desire to multiplex as many disease targets within a single reaction in order to reduce the costs of testing. ESI-MS/MS is a robust and versatile technique with established utilization in research and clinical laboratories. Nevertheless, rarely have attempts been made to adopt this technology for DNA quantification. Liquid chromatography mass spectrometry has been used to indirectly quantify DNA through components such as nucleotides, nucleosides or nucleobases following enzymatic digestion26,28 or acid digestion20. This method involves demanding chromatographic separation, which hampers its utilization in high throughput environments such as NBS. Inductively coupled plasma-mass spectrometry is used to directly quantify DNA through phosphorus content and generate Standard Reference Materials, however instrument and workflow requirements are not yet feasible for clinical laboratories21. To our knowledge, no ESI-MS/MS methods have previously been described to quantify DNA in blood or DBS. In this report, we developed a method to quantify DNA in neonatal DBS specimens. This method utilizes ESI-MS/MS, a technology widely available in NBS laboratories. Our method is based on liberation of adenine from DNA using selective acid-catalyzed depurination conditions that result in nearmaximum release of adenine with minimal release of adenine from RNA22 or ATP. The quantity of DNA is then calculated from adenine as previously described22. In this study, we used m/z 136 > 92 for adenine quantification. This transition corresponds to MH+-NH3-HCN, a significant dissociation product of adenine in ESI-MS/MS. Unlike m/z 136 > 119 which corresponds to loss of ammonia, a common pathway that may be shared with other compounds, the transition we employed is more specific to adenine and reduces the risk of interference from isobars that may be present in the specimen. In contrast to other methods which utilize m/z 136 > 119 for detection20, chromatography was not required in our method due to the higher specificity provided by the m/z 136 > 92 transition. The elimination of chromatographic separation in our method fulfilled the high throughput requirement and was compatible with ESI-MS/MS instrument configuration commonly present in NBS environments. Measurement of DNA quantity allows for optimization of assays that are sensitive to input amount, whether it is critical to the success of the assay or consistency of the results. This is of particular importance in NBS due to the nature of DNA extraction methods that are designed for fast generation of relatively crude DNA extracts from DBS. Outcomes of PCRbased assays are particularly influenced by DNA input amount. Whether they be end-point or real-time assays, each assay will have an optimal input range - excessive input may negatively impact the reaction (e.g. increasing risk of nonspecific amplification), whereas too little input will reduce the yield. In assays where the output is measured against a cutoff to establish a positive versus negative result, either scenario may increase the risk of false positive or negative results. Although not currently common in NBS routines, it is foreseeable that some form of next-generation sequencing will find a place in this clinical laboratory discipline8,28. Successful

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library preparation for next-generation sequencing requires reliable DNA extraction from DBS. Our method is useful when evaluating or comparing different extraction methods as demonstrated by calculating recovery of DNA from DBS using our in-house routine extraction method. Compared to other methods such as fluorescence and UV absorbance, our ESI-MS/MS method is advantageous due to high throughput, sensitivity, specificity, low cost and scalability. One limitation of this method is that it measures total DNA present in the sample regardless of its source. If a sample is contaminated with microbial or viral content, their nucleic acid material will contribute to any measurements made. Another limitation is that degraded and/or fragmented DNA, which otherwise might not be suitable for particular molecular assays, will yield similar measurements as higher molecular weight DNA. However, these limitations are not unique to ESI-MS/MS as they also occur in other methods. In this study, a reference interval for DNA quantity in neonatal DBS was determined. Our experimentally determined interval is in agreement with the theoretical estimation in the literature12,26 as shown in Table 2. In summary, we report a novel approach for DNA quantification in neonatal DBS specimens. The method is based on specific acid-catalyzed release of adenine from DNA followed by ESI-MS/MS analysis. Sample preparation involves an efficient washing step that reduces ion suppression and removes ATP as another source of adenine in our analysis. The specific ion transitions that were employed in this work reduced the risk of interference from adenine isobars and allowed for accurate DNA quantification. Our method provides reliable estimate of the total amount of DNA in DBS specimens. Knowledge of the total amount of DNA originally present in a DBS provides investigators with information on whether this analyte is efficiently recovered in their extraction procedures. The method as described is sensitive, specific, accurate and scalable to high throughput settings and can be applied using equipment currently deployed in NBS laboratories.

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ASSOCIATED CONTENT

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Supporting Information

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Table S-1. Wash solutions tested during method development.

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AUTHOR INFORMATION

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Corresponding Author

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*Tel: [97137137416]; Fax: [9717672022]; Email: [[email protected]].

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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We thank Svetlana Ogrel, Monica Lamoureux, Michael Kowalski and Newborn Screening Ontario technologists for their technical and logistical contribution to this work.

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REFERENCES

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