TY - JOUR
AU - Levin, Barbara, C
AB - Abstract Background: Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G, that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. Methods: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. Results: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the wild-type sequence. Conclusion: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods. A multitude of human mitochondrial DNA (mtDNA) 1 diseases have been correlated with single base pair mutations, insertions, and deletions (1)(2). Most of these diseases involve the neuromuscular system, but deafness, diabetes, epilepsy, progressive dementia, hypoventilation, cardiac insufficiency, renal dysfunction, and sudden onset blindness are other symptoms that have been associated with mtDNA mutations. Because each cell contains multiple mitochondria and each mitochondrion usually contains more than one mtDNA chromosome, heteroplasmy (i.e., the condition in which the mutant mtDNA and wild-type mtDNA coexist in the same individual) is usually present in these disease states. Detection of these heteroplasmies depends on the relative concentrations of the heteroplasmic variations in the sample tissue. Tissues that can be obtained in a noninvasive manner (e.g., blood, hair, and buccal swabs) usually contain a lower percentage of the heteroplasmic mutant DNA than a more highly metabolic tissue such as muscle (3). Among mtDNA disorders, 80% of patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) have the A3243G mutation, the most common mtDNA point mutation detected in an examination of ∼2000 patients suspected of having mtDNA disorders (3)(4). [The numbering system used here is the same as that of the Cambridge Reference Sequence (5)]. However, there are still cases where the phenotypic symptoms are used to diagnose the disease because the heteroplasmic mutation is not detectable by PCR and sequencing (6). These phenotypic symptoms include strokes between the ages of 5 and 15 years, migraine headaches, seizures, ataxia, myoclonus, deafness, retinopathy, dementia, myopathy, kidney disease, myalgia, ophthalmoplegia, cardiomyopathy, cardiac conduction defects, and/or type II diabetes mellitus (7). Detection of low-frequency heteroplasmic mtDNA mutations is difficult, and in many cases it is not possible to distinguish between the absence of the mutation and the inability of the analytical method to detect it in a given tissue (8). Some investigators have reported the inability to detect any mutant DNA in individuals known to have the disease (9)(10). Many laboratories use PCR amplification followed by restriction enzyme analysis or an allele-specific oligonucleotide approach to detect these mutations (3)(8). Prior research in our laboratory, using PCR products that we prepared containing a single base pair heteroplasmy present at concentrations ranging from 1% to 50%, showed that sequencing could detect a heteroplasmy present at 20% but that 10% was indistinguishable from background (11). With these prepared heteroplasmic samples, the addition of a specially designed peptide nucleic acid (PNA) (11)(12)(13) enabled us to detect the heteroplasmy that was present at 5%. In our method, the PNA interferes with extension elongation and, thus, amplification of the wild-type DNA, but allows amplification of the DNA containing the single base pair heteroplasmic mutation. Using this approach with the A3243G MELAS mutation, we found that the single-nucleotide MELAS mutation that is present in patients with very low to undetectable heteroplasmic concentrations (1–20%) becomes the dominant product and readily discernable. Use of the specifically designed PNA, primers, and information on the optimal conditions described here will allow anyone with PCR and sequencing capabilities to easily screen suspected patients and their maternal relatives for the A3243G MELAS mutation. Materials and Methods dna from melas patients and controls The DNA from eight patients diagnosed with MELAS was provided by Georgetown University Medical Center (Washington, DC). The research done at NIST was deemed exempt by the NIST Institutional Review Board because the DNA samples were coded and untraceable to the original donors. The DNA was extracted from peripheral blood leukocytes by the salting out method of Lahiri and Nurnberger (14). DNA samples were reconstituted at 10 ng/μL in buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA). Table 1 shows the percentage of the heteroplasmic mutation in the mtDNA from each patient’s blood estimated by the restriction fragment length polymorphism (RFLP) procedure (15). Table 1. Estimated percentage of the MELAS A3243G mutation in the mtDNA from blood of eight patients. Patient no. . Percentage estimated by RFLP . 1 10 2 20 3 15 4 16 5 3 6 5 7 1 8 9 Patient no. . Percentage estimated by RFLP . 1 10 2 20 3 15 4 16 5 3 6 5 7 1 8 9 Open in new tab Table 1. Estimated percentage of the MELAS A3243G mutation in the mtDNA from blood of eight patients. Patient no. . Percentage estimated by RFLP . 1 10 2 20 3 15 4 16 5 3 6 5 7 1 8 9 Patient no. . Percentage estimated by RFLP . 1 10 2 20 3 15 4 16 5 3 6 5 7 1 8 9 Open in new tab DNA extracted from three cell lines [9947A (from NIST Standardized Reference Material 2392), GM03798 (from Coriell Repository), and HL-60 (from American Type Culture Collection)] that had only A at nucleotide position (np) 3243 were used as wild-type controls. The HL-60 DNA was used to serially dilute (1:10, 1:100, and 1:1000; for the 1:10 dilution, starting with equal concentrations of the MELAS and HL-60 DNA, 2 μL of MELAS sample was mixed with 18 μL of HL-60 DNA; 2 μL of the 1:10 dilution was then diluted in 18 μL of HL-60 DNA for the 1:100 dilution; finally, 2 μL of the 1:100 dilution was then diluted in 18 μL of HL-60 DNA for the 1:1000 dilution) the DNA sample from the MELAS patient with the estimated 1% heteroplasmy to provide samples with even lower proportions of the heteroplasmy to determine the limit of detection. PNAs PNA oligomers are DNA analogs in which the ribose-phosphate backbone is replaced by a neutral backbone consisting of N-(2-aminoethyl)glycine units linked by amide bonds (12). The four bases (A, C, G, and T) are attached to the achiral peptide-like backbone with methylene carbonyl linkages. PNAs mimic DNA, binding to complementary DNA strands but with greater specificity and binding strength. Three PNAs were synthesized for this study (Fig. 1). One pair of PNAs (PNAs 1 and 2; 9-bp long) was designed to bind to both the sense and antisense strands of the wild-type mtDNA with only minimal overlap around np 3243, the site of the MELAS mutation. They were designed in this fashion so that the matched PNA pair (PNAs 1 and 2) could be added at the same time to the PCR mixture to prevent amplification of both strands without binding to each other. PNA 3 (11-bp long) was designed to match the sense sequence (light-strand) and thereby bind to the antisense strand (heavy-strand) with np 3243 in the middle. All of the PNAs were designed to be complementary to the wild-type DNA, not the mutant. Figure 1. Open in new tabDownload slide Binding sites of the PNAs to DNA and its complement. np 3243 is underlined. (A), PNA 3 with the Cambridge Reference Sequence (CRS) and its complement; (B), PNA 3 with the CRS mutant; (C), PNA pair 1 and 2 with the CRS and its complement. In A, B, and C, the 22-oligomer segment of the CRS used in the thermal stability studies and its complement are shown. In the actual experiments, the PNAs were added to the PCR reaction mixture that produced a 586-bp amplicon containing np 3243. Figure 1. Open in new tabDownload slide Binding sites of the PNAs to DNA and its complement. np 3243 is underlined. (A), PNA 3 with the Cambridge Reference Sequence (CRS) and its complement; (B), PNA 3 with the CRS mutant; (C), PNA pair 1 and 2 with the CRS and its complement. In A, B, and C, the 22-oligomer segment of the CRS used in the thermal stability studies and its complement are shown. In the actual experiments, the PNAs were added to the PCR reaction mixture that produced a 586-bp amplicon containing np 3243. The PNA molecules were synthesized on an ABI 433A Peptide Synthesizer (Applied Biosystems) using solid-phase peptide synthesis protocols and tert-butoxycarbonyl (t-Boc) chemistry (16). The PNA oligomers were synthesized from the COOH terminus to the NH2 terminus with the carboxyl end of the first PNA monomer being protected by an attachment to a polystyrene bead. The first monomer was attached to the 4-methylbenzhydrylamine-HCl (MBHA) resin (Applied Biosystems) with use of N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) as a coupling reagent and capped with PNA cap solution (Applied Biosystems) (17). Strand elongation was initiated by simply washing solutions over the beads at appropriate times. The NH2 terminus was temporarily protected by the t-Boc group during the coupling steps. The synthesized PNA was cleaved from the resin with a trifluoroacetic acid–trifluoromethanesulfonic acid reagent and precipitated with ether. The products were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (13). Analysis with a HPLC (Waters Corp.) using a C18 column heated at 323 K to minimize aggregation of the PNAs indicated that the purity of the three PNA molecules was >90%. PNA concentrations in the PCR buffer were determined by ultraviolet absorbance measurements at 260 nm using estimated extinction coefficients of 7.8 × 104, 8.9 × 104, and 11.2 × 104 cm−1 M−1 for PNAs 1, 2, and 3, respectively [the extinction coefficients were calculated with information from Applied Biosystems (18)]. ultraviolet thermal stability measurements of pna/dna duplexes Two 22-oligomer DNA molecules and their 22-oligomer complements (Table 2) were synthesized by Invitrogen/Gibco BRL (Rockville, MD) and used to prepare the wild-type mtDNA/PNA and mutant mtDNA/PNA duplexes for the ultraviolet thermal stability measurements. The total strand concentrations were in the range of 3–5 μmol/L, and the PNA/DNA duplexes were formed by mixing equal concentrations of each strand. To determine the optimum operating temperatures for PCR, the dissociation, or “melting”, temperatures of these DNA/PNA duplexes were determined by the increase in the absorbance (monitored at 260 nm with a PerkinElmer Lambda 4B Spectrophotometer) as the temperature of the duplex solutions was increased. The sample cell containing the duplex solution was heated at a rate of 1 K/min from 20 to 90 °C by means of a thermal electric heater while the reference cell containing just the buffer solution was maintained at room temperature. The results were analyzed by EXAM (19), a software program that normalizes the absorbances to the total absorbance change and fits the normalized data to a two-state transition model to determine the melting temperature (Tm) and dissociation van’t Hoff enthalpy (ΔHv) of the duplex at a known concentration (c) in solution. The Tms of the duplexes at lower concentrations used in the PCR could be determined with use of the known Tm at a given duplex concentration, the ΔHv, and the following equation: \[\ \frac{\mathrm{d}(\mathrm{ln}c)}{\mathrm{d}(1/T_{\mathrm{m}})}\ {=}\ {-}{\Delta}H_{\mathrm{v}}/R\] where d indicates the derivative, and R is the ideal gas constant (8.31451 J mol−1K−1). Table 2. DNA oligomers used in ultraviolet thermal stability measurements. DNA oligomer . Base sequence1 . Wild type 5′-ATTACCGGGCTCTGCCATCTTA-3′ Wild-type complement 5′-TAAGATGGCAGAGCCCGGTAAT-3′ Mutant 5′-ATTACCGGGCCCTGCCATCTTA-3′ Mutant complement 5′-TAAGATGGCAGGGCCCGGTAAT-3′ DNA oligomer . Base sequence1 . Wild type 5′-ATTACCGGGCTCTGCCATCTTA-3′ Wild-type complement 5′-TAAGATGGCAGAGCCCGGTAAT-3′ Mutant 5′-ATTACCGGGCCCTGCCATCTTA-3′ Mutant complement 5′-TAAGATGGCAGGGCCCGGTAAT-3′ 1 np 3243 is underlined and in bold. Open in new tab Table 2. DNA oligomers used in ultraviolet thermal stability measurements. DNA oligomer . Base sequence1 . Wild type 5′-ATTACCGGGCTCTGCCATCTTA-3′ Wild-type complement 5′-TAAGATGGCAGAGCCCGGTAAT-3′ Mutant 5′-ATTACCGGGCCCTGCCATCTTA-3′ Mutant complement 5′-TAAGATGGCAGGGCCCGGTAAT-3′ DNA oligomer . Base sequence1 . Wild type 5′-ATTACCGGGCTCTGCCATCTTA-3′ Wild-type complement 5′-TAAGATGGCAGAGCCCGGTAAT-3′ Mutant 5′-ATTACCGGGCCCTGCCATCTTA-3′ Mutant complement 5′-TAAGATGGCAGGGCCCGGTAAT-3′ 1 np 3243 is underlined and in bold. Open in new tab pcr with and without pna The PCR primers were designed to amplify a 586-bp region that included np 3243. They were the same as primer set 12, which is used in NIST Standardized Reference Material 2392 for sequencing the entire human mtDNA (20). Primer set 12 consists of a forward primer that starts at np 2972 and is 21 oligomers long and a reverse primer that starts at np 3557 and is 20 oligomers long (Table 3). Table 3. Primers used in this study. Primer . Base sequence . F2972 5′-ATAGGGTTTACGACCTCGATG-3′ R3557 5′-AGAAGAGCGATGGTGAGAGC-3′ Primer . Base sequence . F2972 5′-ATAGGGTTTACGACCTCGATG-3′ R3557 5′-AGAAGAGCGATGGTGAGAGC-3′ Open in new tab Table 3. Primers used in this study. Primer . Base sequence . F2972 5′-ATAGGGTTTACGACCTCGATG-3′ R3557 5′-AGAAGAGCGATGGTGAGAGC-3′ Primer . Base sequence . F2972 5′-ATAGGGTTTACGACCTCGATG-3′ R3557 5′-AGAAGAGCGATGGTGAGAGC-3′ Open in new tab The PCR reaction mixture contained 1 μL of DNA, 0.5 μL of 5 U/μL AmpliTaq GoldTM DNA polymerase (final concentration, 2.5 U; Applied Biosystems), 5 μL of GeneAmp 10× PCR Buffer (Applied Biosystems), 1 μL of 10 mM deoxynucleoside triphosphate (dNTP) mixture (final concentration, 0.2 mM each; Invitrogen), 1 μL each of the forward and reverse 10 μM primers (final concentration, 0.2 μM each), 200 μM PNA (in various amounts to examine the effect of PNA concentration; final concentrations ranged from 0.4 to 24 μM), plus H2O to a final volume of 50 μL. The 10× buffer (pH 8.3) contained 100 mM Tris-HCl, 15 mM MgCl2, and 500 mM KCl. To examine the effect of DNA concentration, the DNA concentrations were varied from 0.1 to 10 ng/μL. Thermal cycling was conducted in a PerkinElmer Model 9700 thermocycler. Multiple thermocycle conditions were examined depending on which combination of DNA/PNA was being tested. The most successful combination was DNA/PNA 3 and consisted of an initial incubation step of 95 °C for 10 min (required for AmpliTaq Gold activation), followed by 35 cycles of 15 s at 94 °C (denaturation), 20 s at 77 °C, ramping at 0.6 °C/s (20% rate) to 70 °C for 10 s (annealing PNA to wild-type DNA), ramping at 0.9 °C/s (30% rate) to 56 °C for 70 s (primer annealing and extension), and ending with a final extension of 7 min at 56 °C. A sample of the amplified DNA was examined by electrophoresis in a 2% agarose gel and stained with ethidium bromide to assess the purity and size of the PCR product. The size of the product was estimated by comparison with the gel marker ladder (Research Genetics). Before sequencing, the PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN Inc.). sequencing PCR products were sequenced in both the forward and reverse directions with the same primers as those used for the PCR. Cycle sequencing with fluorescent dye-labeled terminators was performed using an ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase FS (Applied Biosystems). Samples were prepared using 1 μL of primer (initial concentration, 10 μmol/L), 1 μL of DNA, 8 μL of the mixture from the BigDye Terminator Cycle Sequencing Ready Reaction Kit, and 10 μL of distilled water. Thermal cycling was conducted in a PerkinElmer Model 9700 thermocycler and started with 1 min at 96 °C. The reaction then underwent 25 cycles of 96 °C for 15 s (denaturation), 50 °C for 5 s (annealing), and 60 °C for 2 min (extension). The DNA product was purified using Edge Gel Filtration Cartridges (Edge Biosystems). The samples were dried on a RC10.10 Centrifugal Evaporator (Jouan) and reconstituted in 20 μL of Template Suppression Reagent (Applied Biosystems). Sequencing of the fluorescently labeled purified DNA was performed with a ABI PRISM 310 Genetic Analyzer (Applied Biosystems) and a 47 cm × 50 μm capillary column. Data analysis was executed with the Sequence Analysis and Sequence Navigator software packages (Applied Biosystems). estimation of the heteroplasmy concentration by rflp The percentage of the A3243G heteroplasmic concentration was estimated by RFLP. Primers homologous to heavy-strand np 3116–3134 and light-strand np 3353–3333 of the mtDNA were used for PCR amplification of the region containing the A3243G mutation and yielded a PCR product of 238 bp. Digestion of this product with the HaeIII restriction enzyme produced DNA fragments of 169, 37, and 32 bp. The presence of the A3243G mutation generates an additional HaeIII restriction site by cleaving the 168-bp fragment into 97- and 72-bp fragments. The DNA fragments were separated by 12% polyacrylamide gel electrophoresis, and the percentage of the heteroplasmic mutation was estimated by densitometer scanning. Results PNAs Three different PNAs were designed to bind to the wild-type DNA in the region surrounding np 3243, the site of the low-frequency heteroplasmic mtDNA MELAS mutation (Fig. 1). One set of PNA pairs was designed to bind to both the heavy and light mtDNA strands and overlap only around np 3243. Because this pair of PNAs was offset and not complementary to each other, we were hoping that it would inhibit the amplification of both strands of the wild-type mtDNA but not bind to each other. The thermal Tm measurements of the 22-oligomer segment of the wild-type or the mutant DNA bound to the various PNAs indicated that PNA pair 1 and 2 and PNA 3 were good candidates for further study. Determination of the thermal Tms for the PNA/wild-type mtDNA and PNA/mutant mtDNA as well as the optimal conditions for amplification and sequencing was necessary for success. The thermal melting dissociation curves for the successful PNA 3/wild-type mtDNA and PNA 3/mutant mtDNA duplexes are shown in Fig. 2. Table 4 shows the thermal Tms that were found for each PNA/DNA duplex used in the final experiments and the dissociation ΔHv values needed for the determination of the Tms of the duplexes at the lower concentrations used in the PCR reactions (see Materials and Methods). Figure 2. Open in new tabDownload slide Ultraviolet melting measurements on PNA3/DNA (Mutant) and on PNA 3/DNA (WT). Note the increase in the thermal stability of the PNA3/DNA (wild-type) duplex (Tm = 77 °C; ΔHv = 1101 kJ/mol) relative to the PNA3/DNA (mutant) duplex (Tm = 68 °C; ΔHv = 875 kJ/mol). The total strand concentration in both samples was 3 μmol/L. Figure 2. Open in new tabDownload slide Ultraviolet melting measurements on PNA3/DNA (Mutant) and on PNA 3/DNA (WT). Note the increase in the thermal stability of the PNA3/DNA (wild-type) duplex (Tm = 77 °C; ΔHv = 1101 kJ/mol) relative to the PNA3/DNA (mutant) duplex (Tm = 68 °C; ΔHv = 875 kJ/mol). The total strand concentration in both samples was 3 μmol/L. Table 4. PNA/DNA Tms and dissociation enthalpy values. PNA no. . Base sequence . PNA/wild-type mtDNA . . PNA/mutant mtDNA . . . . Tm, °C . ΔHV, kJ/mol . Tm, °C . ΔHV, kJ/mol . 1 5′-CTCTGCCAT-3′ 76 2295 29 1168 2 5′-GAGCCCGGT-3′ 71 1365 43 856 3 5′-GGCAGAGCCCG-3′ 77 1101 68 875 PNA no. . Base sequence . PNA/wild-type mtDNA . . PNA/mutant mtDNA . . . . Tm, °C . ΔHV, kJ/mol . Tm, °C . ΔHV, kJ/mol . 1 5′-CTCTGCCAT-3′ 76 2295 29 1168 2 5′-GAGCCCGGT-3′ 71 1365 43 856 3 5′-GGCAGAGCCCG-3′ 77 1101 68 875 Open in new tab Table 4. PNA/DNA Tms and dissociation enthalpy values. PNA no. . Base sequence . PNA/wild-type mtDNA . . PNA/mutant mtDNA . . . . Tm, °C . ΔHV, kJ/mol . Tm, °C . ΔHV, kJ/mol . 1 5′-CTCTGCCAT-3′ 76 2295 29 1168 2 5′-GAGCCCGGT-3′ 71 1365 43 856 3 5′-GGCAGAGCCCG-3′ 77 1101 68 875 PNA no. . Base sequence . PNA/wild-type mtDNA . . PNA/mutant mtDNA . . . . Tm, °C . ΔHV, kJ/mol . Tm, °C . ΔHV, kJ/mol . 1 5′-CTCTGCCAT-3′ 76 2295 29 1168 2 5′-GAGCCCGGT-3′ 71 1365 43 856 3 5′-GGCAGAGCCCG-3′ 77 1101 68 875 Open in new tab pcr PCR reactions were conducted in the presence of PNA pair 1 and 2 or PNA 3, using thermocycler conditions tailored to each (the Materials and Methods provides only those conditions that were successful). Reactions were performed with concentrations of 0, 0.4, 1, 2, 4, 8, 16, and 24 μmol/L of each PNA. Two percent agarose gels of the PCR products with and without the PNAs in the PCR reaction mixture showed single strong bands of the expected amplicon size. Fig. 3A shows the agarose gel bands after electrophoresis of the PCR products from a representative MELAS sample over the range of PNA concentrations. Figure 3. Open in new tabDownload slide Representative 2% agarose gels of PCR products with various PNA 3 concentrations (0–24 μmol/L). The Gel Marker Ladder (lane S) indicates that the size of the products is consistent with the expected size of 586 bp. The negative control (lane C) contains no DNA. (A), DNA from a MELAS blood sample showing successful amplification at all PNA concentrations. (B), DNA from a control wild-type blood sample (no MELAS mutation) showing inhibition of amplification at all PNA concentrations with the greatest inhibition at ∼4 μmol/L PNA. Figure 3. Open in new tabDownload slide Representative 2% agarose gels of PCR products with various PNA 3 concentrations (0–24 μmol/L). The Gel Marker Ladder (lane S) indicates that the size of the products is consistent with the expected size of 586 bp. The negative control (lane C) contains no DNA. (A), DNA from a MELAS blood sample showing successful amplification at all PNA concentrations. (B), DNA from a control wild-type blood sample (no MELAS mutation) showing inhibition of amplification at all PNA concentrations with the greatest inhibition at ∼4 μmol/L PNA. PCR reactions were also performed on the control DNA samples under conditions identical to those used for the MELAS samples. Fig. 3B shows the agarose gel of the PCR products from the control HL-60 DNA over the same range of PNA concentrations as above. The concentration effect of PNA (which was designed to inhibit the wild-type nucleotide) is clearly indicated and shows a maximum inhibition of the PCR at 4 μmol/L. sequence analysis of pcr products Subsequent sequencing of the PCR products indicated that regardless of PNA concentration or a wide range of thermocycle conditions, the presence of PNA pair 1 and 2 during PCR had little or no effect on the wild-type/mutant ratio seen in the absence of PNA. However, PNA 3, which was designed to bind to the heavy strand with np 3243 in the middle, successfully inhibited the amplification of wild-type mtDNA while allowing the mutant mtDNA to amplify. Both the forward and reverse sequences of the PCR products generated in the presence of PNA 3 (0.4–24 μmol/L) showed that the MELAS G mutation (reverse C) at np 3243 was now the dominant species over the entire range of PNA concentrations. However, we found that a PNA concentration of ∼2 μmol/L was optimal for the greatest suppression of the adenine nucleotide (the wild-type mtDNA; Fig. 4). Figure 4. Open in new tabDownload slide Sequence analysis of a representative MELAS sample amplified with no PNA or various concentrations of PNA 3 (0.4, 1, 2, 4, 8, 16, or 24 μmol/L). In the absence of PNA 3, the MELAS (G) mutation is barely visible above the baseline noise and the wild type (A) is the dominant species. In the presence of PNA 3, the MELAS (G) mutation at np 3243 is preferentially amplified and is the dominant species at all PNA concentrations, although the optimal concentration to suppress the amplification of the wild-type A was 2 μmol/L. Figure 4. Open in new tabDownload slide Sequence analysis of a representative MELAS sample amplified with no PNA or various concentrations of PNA 3 (0.4, 1, 2, 4, 8, 16, or 24 μmol/L). In the absence of PNA 3, the MELAS (G) mutation is barely visible above the baseline noise and the wild type (A) is the dominant species. In the presence of PNA 3, the MELAS (G) mutation at np 3243 is preferentially amplified and is the dominant species at all PNA concentrations, although the optimal concentration to suppress the amplification of the wild-type A was 2 μmol/L. As seen in Fig. 5, in the absence of PNA, it was difficult if not impossible to detect the presence of the mutation because it appeared to be totally absent or indistinguishable from the baseline noise in our MELAS samples obtained from blood. But, as seen in Fig. 4, in the presence of PNA 3, the mutant peak becomes the predominant peak in the mtDNA. This was true for all eight MELAS patients. Figure 5. Open in new tabDownload slide Sequence electropherograms of two MELAS patient samples amplified in the absence of PNA, showing the difficulty of detecting the A3243G mutation. (a), representative sample of six of the eight samples shows a small heteroplasmic guanine mutant peak (G), barely distinguishable from baseline noise. (b), representative sample of two of eight patient samples in which no guanine mutant peak was detectable. Figure 5. Open in new tabDownload slide Sequence electropherograms of two MELAS patient samples amplified in the absence of PNA, showing the difficulty of detecting the A3243G mutation. (a), representative sample of six of the eight samples shows a small heteroplasmic guanine mutant peak (G), barely distinguishable from baseline noise. (b), representative sample of two of eight patient samples in which no guanine mutant peak was detectable. determination of lowest detectable heteroplasmy The MELAS sample estimated to have the lowest percentage of the mutant heteroplasmy (1%) was diluted 10-, 100-, and 1000-fold with control DNA from HL-60 and amplified in the presence of PNA concentrations of 0, 0.4, 1, 2, 4, 8, 16, and 24 μmol/L. The guanine heteroplasmy was clearly visible in the samples diluted to a final heteroplasmic concentration of 0.1% and amplified in the presence of PNA, although it was no longer the dominant peak (Fig. 6). At the greater dilutions, the heteroplasmic mutation was not visible regardless of the concentration of PNA. Figure 6. Open in new tabDownload slide Sequence showing the MELAS heteroplasmic mutation, after PCR in the presence of 2 μmol/L PNA, of the patient’s mtDNA that was diluted 10-fold with control DNA. Fig. 6 indicates that the limit of detection of the heteroplasmy under these conditions in the presence of PNA 3 is 0.1%. The heteroplasmy is clearly visible but no longer the dominant peak. Figure 6. Open in new tabDownload slide Sequence showing the MELAS heteroplasmic mutation, after PCR in the presence of 2 μmol/L PNA, of the patient’s mtDNA that was diluted 10-fold with control DNA. Fig. 6 indicates that the limit of detection of the heteroplasmy under these conditions in the presence of PNA 3 is 0.1%. The heteroplasmy is clearly visible but no longer the dominant peak. effect of dna concentration In addition to varying the concentration of PNA added to the PCR reaction, we varied the amount of DNA added to the PCR reaction by 100-fold (0.1, 1, or 10 ng/μL; final concentrations, 2, 20, or 200 μmol/L) to assure that the results of our PCR-PNA reactions were not sensitive to the DNA concentration. Regardless of whether the final concentration was 2, 20, or 200 μmol/L DNA in the PCR reaction mixture, the results were the same: PNA 3 effectively blocked amplification of the wild type while allowing amplification of the mutant species (data not shown). Discussion MELAS is a devastating disease that causes seizures, ataxia, psychomotor regression, hemiparesis/hemianopia, cortical blindness, migraine-like headaches, dystonia, muscle weakness, exercise intolerance, sensorineural hearing loss, lactic acidosis, ragged-red fibers in the muscles, and short stature (21). Some patients also exhibit myoclonus, peripheral neuropathy, cardiac conduction defects, cardiomyopathy, and Fanconi syndrome. Some diabetes mellitus patients also have the same mtDNA A3243G mutation found in MELAS patients (21). The A3243G mutation occurs in the dihydrouridine loop in tRNALeu(UUR) (7). The finding that this mutation is also in the middle of the transcription terminator binding site and may perturb transcription has been proposed as a possible mechanism for the multiple symptoms associated with MELAS. Wallace et al. (7) discussed the evidence that both support and refute this proposed mechanism. However, the finding that 10 other single base pair mutations (6 of which are found outside the transcription terminator binding site, and of those, 2 have been associated with MELAS) are also in this tRNA and have been correlated with seven different mitochondrial diseases indicates that there probably are additional factors that need to considered in deciphering the mechanism whereby this mutation causes MELAS. Eighty percent of MELAS patients have the heteroplasmic mtDNA A3243G mutation in which adenine is the wild-type base and guanine is the mutation associated with the disease. This mutation is usually present in low amounts compared with the wild type, especially in tissues that are obtained in a relatively noninvasive manner (e.g., blood, hair, or buccal swabs; Fig. 5). The question arises as to whether the 20% of the MELAS patients without a detectable mutation actually have the mutation but at a concentration that is not detectable in that particular tissue with current technologies. There have been cases reported in which a single heteroplasmic base pair mutation was readily found in muscle but was undetectable in blood (22)(23), or where the A3243G mutation was detected in muscle and hair follicles in 10 individuals but detected in the blood of only 5 of those individuals (24). These 10 patients ranged in age from 23 to 73 years, and the mutation was found in the blood of the younger patients (23, 28, 38, 41, and 45 years of age), but not in the older patients (47, 58, 62, 62, and 73 years of age). Weber et al. (23), however, took muscle biopsies over a 12-year period and found that the mutation increased with time and that the increase correlated with an increase in clinical symptoms. Other investigators have also found that this mutation decreases in the blood after birth at a mean rate estimated at ∼0.69% ± 0.61% per year (25) or 1.4% per year (26). A recent study used PNAs to examine specific mtDNA mutations in brain and muscle tissue from various age groups and found that the MELAS and MERRF mutations did not accumulate with age, but mutations at np 414 adjacent to the control region promoters did accumulate with age in muscle but not in brain (27). Therefore, mutations seem to increase in the muscle but decrease in the blood over time, a circumstance that may make the detection of heteroplasmic mutations in blood samples more difficult in the elderly. PNAs have been used to detect polymorphisms and mutations primarily by preventing primer annealing during PCR (27)(28)(29)(30). Some investigators have used free solution capillary electrophoresis in which PNA/DNA duplexes are analyzed at high temperatures in the presence of denaturants (31)(32). This analysis requires separate runs to distinguish the mutant and wild type. In these types of experiments, buffer composition and increased column temperatures are important in controlling the hybridization, and the PNA/DNA duplexes are noted by the shift in mobility from the unhybridized oligonucleotide. In these cases, the investigators were examining mutations in nuclear DNA so that heteroplasmy was not an issue, although Perry-O’Keefe et al. (32) were able to detect an individual known to be heterozygous for the cystic fibrosis mutation. Butler et al. (13) showed that MALDI-TOF MS could be used to characterize PNA oligomers, and Ross et al. (33) made PNA probes that were hybridized to single-stranded PCR biotinylated DNA products that were immobilized on streptavidin-coated magnetic beads. The biotinylated PCR product was constructed by labeling the reverse primer with 5′ biotin. These immobilized complexes were analyzed by MALDI-TOF MS. These experiments were designed to recognize homoplasmic polymorphisms, but not heteroplasmic polymorphisms or low-frequency heteroplasmic mutations. Therefore, based on previous work (11) in our laboratory that showed that specifically designed PNAs could interfere with PCR of the wild-type DNA while allowing amplification of the heteroplasmic mutant DNA, we have developed an easy method for the unequivocal detection of the specific MELAS A3243G mutation when it is present in blood samples at ≥0.1% and at proportions that are not detectable by typical DNA sequencing (≤20%). The question arose as to what would happen if there were a polymorphism other than or in addition to np 3243 in the PNA binding area. If the other polymorphism were a low-frequency heteroplasmic mutation, the PNA would still bind to the wild-type DNA, preventing its amplification, and the other mutation would be detectable when the amplicon was sequenced. If the polymorphism existed along with the np 3243 mutation, then both should become detectable when sequenced. Only if another polymorphism or mutation existed in the PNA binding region and were homoplasmic (all the mtDNA contains the change), then the PNA would not bind and the low-frequency heteroplasmy at np 3243 would be as difficult to detect as it currently is. However, if the polymorphism was homoplasmic or a high-frequency heteroplasmy, it should not be difficult to detect by PCR and sequencing. Therefore, if one detects a homoplasmic polymorphism or a high-frequency heteroplasmy within the PNA binding site (np 3238–3248), it should alert the investigator that further studies are needed to detect whether the low-frequency MELAS mutation exists. In addition, a search of the MITOMAP web site (34) indicated that their extensive literature search of mtDNA polymorphisms has not detected any polymorphisms (other than np 3243) at any of the nucleotide sites covered by this PNA (np 3238–3248). We therefore believe that this will be a rare problem, if a problem at all. In this study, sequencing of mtDNA samples from eight MELAS patients showed the presence of a minor heteroplasmic guanine component at np 3243 at concentrations that were nearly indistinguishable from the background noise in six of the samples and that were not observable at all in two of the samples (Fig. 5). The method presented here, PCR under the described thermal cycling conditions in the presence and in absence of our specific PNA, followed by mtDNA sequence analysis, provided definitive evidence for the presence of the MELAS (A3243G) heteroplasmy. This method is sensitive enough to detect the heteroplasmy in blood samples at 0.1% so that investigators do not have to perform more invasive and painful muscle or other tissue biopsies. It is hoped that the enhanced detection method described here will enable investigators to find the MELAS mutation in both asymptomatic carriers and symptomatic patients in whom the mutation without the presence of PNA is not detectable. Because MELAS and many mtDNA diseases are age-dependent (with increasing age, the mutant mtDNA reaches a threshold and the disease becomes symptomatic), we hope that sometime in the future the ability to detect the mutation in asymptomatic carriers may permit therapeutic intervention before the disease reaches threshold values. This PNA binding method will also be helpful in prenatal diagnosis and preimplantation genetic diagnosis. The problem today is that the difficulty in detecting the mutation can interfere with obtaining an accurate diagnosis, providing correct genetic counseling, and devising the correct therapeutic approach. One option currently available to prevent the genetic transmission of mtDNA mutations is the use of donor oocytes. A future option may be transfer of the nucleus of the mother to a donor oocyte with wild-type mtDNA (35). Thorburn and Dahl (35) caution not to use a maternal relative as the oocyte donor because her oocytes may carry the mutation although her blood has no detectable mutant mtDNA. The method described here will allow investigators to feel confident that the blood is providing the correct result. PNAs have been used to detect mutations in several other studies. However, in contrast to most of this research using PNA to detect mutations (27)(28)(29)(30), our PNA binding site is in the middle of the amplicon and distant from both the forward and reverse primers. Taylor et al. (36)(37) and Chinnery et al. (38) are examining the possible use of PNAs to inhibit the mutant mtDNA in diseased individuals and allow only the wild-type DNA to replicate. If successful, this approach could lead to a therapeutic solution. However, there are many obstacles to overcome, e.g., getting the PNA into the tissues, cells, and mitochondria; binding the PNA to the mtDNA; and inhibiting the replication of the mutant but not the wild-type mtDNA. There has been some success in delivery of the PNAs to cells grown in culture (38), but it has not been shown that this approach alters the proportions of the active mutation. Because this same mutation, A3243G, is the most common mtDNA mutation associated with type II diabetes mellitus, we are extending our studies to determine whether this sensitive method will allow enhanced detection of the mutation in diabetic patients as well. The methodology described here concerning a specifically designed PNA, primers, and the optimal conditions for PCR and sequencing will allow investigators to detect the A3243G mutation associated with the human mitochondrial disease MELAS even when the heteroplasmic mutation is present in such low concentrations as to be undetectable by other methods. If we determine that this PNA will also detect the previously undetectable mutation in diabetic patients and their maternal relatives, then this methodology may also be useful to the clinical community trying to diagnose type II diabetes mellitus and provide counseling to potential genetic carriers of the disease. " This paper is a contribution of the US National Institute of Standards and Technology (NIST) and is not subject to copyright. Certain commercial equipment, instruments, materials, or companies may be identified in this paper to specify the experimental procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the materials or equipment identified are the best available for this purpose. 1 " Nonstandard abbreviations: mtDNA, mitochondrial DNA; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; PNA, peptide nucleic acid; RFLP, restriction fragment length polymorphism; np, nucleotide position; and MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. References 1 Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases?. Science 1992 ; 256 : 628 -632. Crossref Search ADS PubMed 2 Tully LA, Levin BC. Human mitochondrial genetics. Harding SE eds. Biotechnology and genetic engineering reviews 2000 ; Vol. 17 : 147 -177 Intercept Ltd. Andover, United Kingdom. . Crossref Search ADS 3 Wong L-JC, Senadheera D. Direct detection of multiple point mutations in mitochondrial DNA. Clin Chem 1997 ; 43 : 1857 -1861. Crossref Search ADS PubMed 4 Liang MH, Wong LJC. Yield of mtDNA mutation analysis in 2000 patients. Am J Med Genet 1998 ; 77 : 395 -400. Crossref Search ADS PubMed 5 Anderson S, Bankier AT, Barrell BG, deBrujin MHL, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature 1981 ; 290 : 457 -465. Crossref Search ADS PubMed 6 Koga Y, Fukiyama R, Matsuoka H, Akita Y, Imaizumi T, Kato H. Endothelial dysfunction in patients with MELAS: role of oxidative stress to stroke [Abstract]. Proceedings of the Mitochondria 2001 Meeting, Feb. 28–March 4, 2001, San Diego, CA.. 7 Wallace DC, Brown MD, Lott MT. Mitochondrial genetics. Rimoin DL Connor JM Pyeritz RE eds. Emery and Rimoin’s principles and practice of medical genetics, 3rd ed 1997 ; Vol. 1 : 277 -332 Churchill Livingstone New York. . 8 Wong L-JC, Lam C-W. Alternative, noninvasive tissues for quantitative screening of mutant mitochondrial DNA. Clin Chem 1997 ; 43 : 1241 -1243. Crossref Search ADS PubMed 9 Hammans S, Sweeney M, Brockington M, Morgan-Hughes J, Harding A. Mitochondrial encephalopathies: molecular genetic diagnosis from blood samples. Lancet 1991 ; 337 : 1311 -1313. Crossref Search ADS PubMed 10 Ciafaloni E, Ricci E, Shanske S, Moraes CT, Silvestri G, Hirano M, et al. MELAS: clinical features, biochemistry, and molecular genetics. Ann Neurol 1992 ; 31 : 391 -398. Crossref Search ADS PubMed 11 Tully LA, Schwarz FP, Levin BC. Development of a heteroplasmic mitochondrial DNA standard reference material for detection of heteroplasmy and low frequency mutations. Proceedings of the 10th International Symposium in Human Identification, September 29–October 2, 1999, Orlando, FL. http://www.promega.com/geneticidproc/ussymp10proc/abstracts/24Tully.pdf (Accessed June 18, 2002).. 12 Rose DJ. Characterization of antisense binding properties of peptide nucleic acids by capillary gel electrophoresis. Anal Chem 1993 ; 65 : 3545 -3549. Crossref Search ADS PubMed 13 Butler JM, Jiang-Baucom P, Huang M, Belgrader P, Girard J. Peptide nucleic acid characterization by MALDI-TOF mass spectrometry. Anal Chem 1996 ; 68 : 3283 -3287. Crossref Search ADS PubMed 14 Lahiri DK, Nurnberger JI, Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991 ; 19 : 5444 . Crossref Search ADS PubMed 15 Levin BC, Cheng H, Kline MC, Redman JW, Richie KL. A review of the DNA standard reference materials developed by the National Institute of Standards and Technology. Fresenius J Anal Chem 2001 ; 370 : 213 -219. Crossref Search ADS PubMed 16 Koch T, Hansen HF, Andersen P, Larsen T, Batz HG, Otteson K, et al. Improvements in automated PNA synthesis using Boc/Z monomers. J Peptide Res 1997 ; 49 : 80 -88. 17 Egholm M, Casale RA. The chemistry of peptide nucleic acids. Kates SA Albericio F eds. Solid-phase synthesis 2000 : 549 -578 Marcel Dekker New York. . 18 Applied Biosystems. PNA chemistry for the ExpediteTM 8900 nucleic acid synthesis system; users guide. http://docs.appliedbiosystems.com/pebiodocs/00601308.pdf (Accessed June 18, 2002).. 19 Kirchhoff WH. Exam: a two-state thermodynamic analysis program. NIST Technical Note 1401 1993 : 1 -103 NIST Gaithersburg, MD. . 20 Levin BC, Cheng H, Reeder DJ. A human mitochondrial DNA Standard Reference Material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 1999 ; 55 : 135 -146. Crossref Search ADS PubMed 21 DiMauro S, Schon EA. Mitochondrial DNA mutations in human diseases. Am J Med Genet 2001 ; 106 : 18 -26. Crossref Search ADS PubMed 22 Moraes CT, Ciacci F, Bonilla E, Ionasescu V, Shon EA, DiMauro S. A mitochondrial tRNA anticodon swap associated with a muscle disease. Nat Genet 1993 ; 4 : 284 -288. Crossref Search ADS PubMed 23 Weber K, Wilson JN, Taylor L, Brierley E, Johnson MA, Turnbull DM, et al. A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle. Am J Hum Genet 1997 ; 60 : 373 -380. PubMed 24 Sue CM, Quigley A, Katsabanis S, Kapsa R, Crimmins DS, Byrne E, et al. Detection of MELAS A3243G point mutation in muscle, blood, and hair follicles. J Neurol Sci 1998 ; 161 : 36 -39. Crossref Search ADS PubMed 25 t’Hart LM, Jansen JJ, Lemkes HH, de Knijff P, Maassen JA. Heteroplasmy levels of a mitochondrial gene mutation associated with diabetes mellitus decrease in leucocyte DNA upon aging. Hum Mutat 1996 ; 7 : 193 -197. Crossref Search ADS PubMed 26 Rahman S, Poulton J, Marchington D, Suomalainen A. Decrease of 3243 A→G mtDNA mutation from blood in MELAS syndrome: a longitudinal study. Am J Hum Genet 2001 ; 68 : 238 -240. Crossref Search ADS PubMed 27 Murdock DG, Christacos NC, Wallace DC. The age-related accumulation of a mitochondrial DNA control region mutation in muscle, but not brain, detected by a sensitive PNA-directed PCR clamping based method. Nucleic Acids Res 2000 ; 28 : 4350 -4355. Crossref Search ADS PubMed 28 Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res 1993 ; 21 : 5332 -5336. Crossref Search ADS PubMed 29 Thiede C, Bayerdorffer E, Blasczyk R, Wittig B, Neubauer A. Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated clamping. Nucleic Acids Res 1996 ; 24 : 983 -984. Crossref Search ADS PubMed 30 Kyger EM, Krevolin MD, Powell MJ. Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping. Anal Biochem 1998 ; 260 : 142 -148. Crossref Search ADS PubMed 31 Carlsson C, Jonsson M, Norden B, Dulay MT, Zare RN, Noolandi J, et al. Screening for genetic mutations. Nature 1996 ; 380 : 207 . PubMed 32 Perry-O’Keefe H, Yao X-W, Coull JM, Fuchs M, Egholm M. Peptide nucleic acid pre-gel hybridization: an alternative to Southern hybridization. Proc Natl Acad Sci U S A 1996 ; 93 : 14670 -14675. Crossref Search ADS PubMed 33 Ross PL, Lee K, Belgrader P. Discrimination of single-nucleotide polymorphisms in human DNA using peptide nucleic acid probes detected by MALDI-TOF mass spectrometry. Anal Chem 1997 ; 69 : 4197 -4202. Crossref Search ADS PubMed 34 MITOMAP: a human mitochondrial genome database. http://www.mitomap.org (Accessed July 23, 2002).. 35 Thorburn DR, Dahl HM. Mitochondrial disorders: genetics, counseling, prenatal diagnosis and reproductive options. Am J Med Genet 2001 ; 106 : 102 -114. Crossref Search ADS PubMed 36 Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN. Selective inhibition of mutant mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet 1997 ; 15 : 212 -215. Crossref Search ADS PubMed 37 Taylor RW, Chinnery PF, Turnbull DM, Lightowlers RN. In vitro modification of mitochondrial function. Hum Reprod 2000 ; 15 : 79 -85. Crossref Search ADS PubMed 38 Chinnery PF, Taylor RW, Diekert K, Lill R, Turnbull DM, Lightowlers RN. Peptide nucleic acid delivery into human mitochondria. Gene Ther 1999 ; 6 : 1919 -1928. Crossref Search ADS PubMed © 2002 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Design and Use of a Peptide Nucleic Acid for Detection of the Heteroplasmic Low-Frequency Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS) Mutation in Human Mitochondrial DNA
JF - Clinical Chemistry
DO - 10.1093/clinchem/48.12.2155
DA - 2002-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/design-and-use-of-a-peptide-nucleic-acid-for-detection-of-the-nypC4lnj68
SP - 2155
VL - 48
IS - 12
DP - DeepDyve
ER -