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Detection and Quantification of Heteroplasmic Mutant Mitochondrial DNA by Real-Time Amplification Refractory Mutation System Quantitative PCR Analysis: A Single-Step Approach

¹ Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC.

^aAddress correspondence to this author at: Institute for Molecular and Human Genetics, Georgetown University Medical Center, M4000, 3800 Reservoir Rd. NW, Washington, DC 20007. Fax 202-444-1770; e-mail wonglj{at}georgetown.edu.

	Abstract

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Background: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation.

Methods: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences.

Results: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR–restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations.

Conclusions: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.

	Introduction

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The mitochondrial genome is a circular double-stranded 16 569-bp DNA that encodes 13 protein subunits of the enzyme complexes in the oxidative phosphorylation pathway, the 12S and 16S ribosomal RNAs, and all of the 22 transfer RNAs required for mitochondrial protein synthesis (1)(2). Mitochondrial genetics feature several unique characteristics (3)(4)(5), including non-Mendelian maternal inheritance, a genome with a 10–20 times higher mutation rate than nuclear DNA, and hundreds to thousands of mitochondria per cell and 2–10 copies of circular double-stranded mitochondrial genome per mitochondrion. Therefore, pathogenic mitochondrial DNA (mtDNA)¹ mutations often are in the heteroplasmic state, i.e., coexistence of wild-type and mutant mtDNA. Clinical manifestation of mitochondrial disease is heterogeneous because of the tissue threshold and the distribution of mutant mtDNA among different tissues.

Numerous nucleotide substitutions responsible for respiratory chain defects have been identified (2)(5)(6)(7). The most common point mutation is the A3243G mutation in tRNA leu(UUR), which is responsible for mitochondrial encephalopathy lactic acidosis, and stroke-like episodes (MELAS) syndrome and maternally inherited diabetes and deafness (MIDD). The proportion of mutant mitochondria must reach the threshold to cause clinical phenotype. Thus, to predict a patient’s clinical outcome, it is important to determine the extent of mutant load in the affected or relevant tissue. In the case of MIDD, patients usually carry low percentages of mutant mtDNA in the blood that may not be detected by conventional restriction fragment length polymorphism (RFLP) analysis. This becomes a problem, especially when the determination of carrier status is important to confirm the diagnosis of a maternally inherited mitochondrial disorder and to provide appropriate genetic counseling. The detection limit for RFLP–ethidium bromide (EtBr) gel analysis is 5–10%, and that for radioactive allele-specific oligonucleotide (ASO) dot-blot analysis is 2% (8)(9). However, ASO analysis alone cannot be used to determine the percentage of mutant heteroplasmy.

We recently developed a real-time quantitative PCR (RT-qPCR) that uses allele-specific TaqMan probes to measure the percentage of heteroplasmic A3243G mutation (10), Although the results were consistent with the results from RFLP analysis, because of nonspecific binding of the mutant TaqMan probe to wild-type target DNA sequences and vice versa, the heteroplasmy showed a significant background (unpublished observation). Thus, it was not of value in the measurement of low proportions of mutant heteroplasmy.

To measure low proportions of mutant heteroplasmy, we designed mismatched primers for an amplification refractory mutation system (ARMS) assay. The introduction of one or two mismatched nucleotides immediately 5' to the mutation site greatly increased the binding specificity of the allele-specific modified primers toward either the wild-type or the mutant sequence targets (11). Here we report the validation of the RT ARMS-qPCR assay for the detection and quantification of known DNA samples with the A3243G mutation. The results demonstrate that the RT ARMS-qPCR assay is a rapid, sensitive, reliable, and cost-effective method with potential for use in the detection and quantification of heteroplasmic mtDNA mutants.

	Materials and Methods

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patients, dna samples, and dna isolation
Patients were referred to the Molecular Genetics Laboratory at the Institute for Molecular and Human Genetics, Georgetown University Medical Center, for the mutational evaluation of mitochondrial disorders. Total DNA was isolated from peripheral blood lymphocytes by a salting-out method (12). DNA from muscle was extracted by use of proteinase K digestion followed by standard phenol–chloroform extraction and ethanol precipitation (13). Mutations in mtDNA were analyzed by multiplex PCR–ASO analysis (8)(9). Thirty-three samples testing positive for A3243G and 3 samples testing negative for the mutation were included for RT ARMS-qPCR analysis and were reevaluated by PCR–RFLP analysis.

primers for arms
In ARMS PCR, the reverse primer (5'-TGGCCATGGGTATGTTGTTA-3') was at nucleotide positions 3319–3300 for the amplification of both wild-type mtDNA and the A3243G mutation. The forward primers for RT ARMS-PCR analysis of the A3243G mutation are listed in Table 1 . The numbers correspond to the nucleotide positions in GenBank accession no. NC001807 and the MITOMAP database (2). Originally, a mismatch at the penultimate nucleotide position of the mutation site was introduced in the forward primers (Table 1 , set A) to increase the specificity of the ARMS reaction based on the principles described by Newton et al. (11). Nevertheless, the specificity was not sufficient, and there were background signals at 0% mutant. Two mismatches at the two nucleotides immediately 5' to the mutation site were then introduced (Table 1 , set B), which greatly improved the specificity. Thus, modified forward primers from set B were used for this study. At the mutation site, the wild-type primer containing an "A" would perfectly match the wild-type target sequence but would be a weak "AC" mismatch with the mutant target sequence. Similarly, the mutant primer containing a "G" would be a perfect match with the mutant target sequence but would be a weak "GT" mismatch with the wild-type target sequence. Therefore, introduction of a strong CC mismatch at the penultimate nucleotide is expected to increase the primer specificity (11).

RT-QPCR
The RT ARMS-qPCR assay was performed in triplicate for each wild-type and mutant target sequence. The 20-µL PCR reaction mixture contained 1x Platinum SYBR Green qPCR SuperMix UDG (Invitrogen), 500 nM each primer, Rox dye, and 4 ng of total genomic DNA extract, according to the manufacture’s instructions. Real-time PCR conditions were 2 min at 50 °C and 10 min at 95 °C, followed by 45 cycles of denaturation for 15 s at 95 °C and annealing/extension for 60 s at 63 °C. SYBR Green dye binds to the minor groove of double-stranded DNA and increases the intensity of the fluorescent emissions while the amplicons are produced in each amplification cycle. The fluorescent signal intensities were recorded and analyzed during PCR in an ABI Prism 7700 sequence detector system (Applied Biosystems) using the SDS (Ver. 1.91) software. Dissociation curves for the amplicons were generated after each run to confirm that the increased fluorescence intensities were not attributable to nonspecific signals (primer-dimers). The increase in fluorescent signal is associated with an exponential growth of PCR product during the linear-log phase. The threshold cycle (C_T) is the cycle at which a significant increase in the reaction product is first detected. The higher the initial amount of DNA, the sooner accumulated product is detected in the PCR process and the lower the C_T value. Thus, the C_T values within the linear exponential increase phase are used to measure the original DNA template copy numbers and to construct the calibration curve. If a sample contained >100 000 or <100 copies based on the C_T, the assay was repeated at a higher or lower dilution of the DNA extract so that the measurement would fall within a linear DNA copy number range.

preparation of dna for calibration curves
DNA for the wild-type and mutant target sequences was generated from cloned plasmid DNA containing pCR2.1-TOPO vector (Invitrogen) and PCR products of primers mtF3212 and mtR3471. The copy numbers of the wild-type and mutant DNA sequences were calculated based on the size and molecular weight of the plasmid DNA. Serial dilutions were made, and RT ARMS-qPCR reactions were performed to construct the calibration curves for the wild-type and mutant A3243G DNA sequences (Fig. 1 ).

View larger version (81K): [in this window] [in a new window]   Figure 1. ASO and RFLP analysis of the A3243G heteroplasmic mutation. Corresponding samples 1–36 were analyzed by radioactive ASO (A) and RFLP (B) methods. Rows I, II, and III contain samples 1–12, 13–24, and 25–36, respectively.

measurement of mutant heteroplasmy
Calibration curves for both wild-type and mutant mtDNA were always included in each run. The copy number of the target sequence in the sample was calculated from the C_T number and the calibration curve. The proportion of mutant A3243G sequence was calculated from the copy number of the wild-type and mutant sequences. Alternatively, the proportion of the mutant mtDNA could be calculated from C_T (C_T^wild-type – C_T^mutant), using the formula: Proportion of mutant = 1/(1 + 1/2^C_T).

rflp analysis with agarose gel electrophoresis–ETBRstaining
For RFLP analysis, the primers used were mtF3085 and mtR3406, which produced a PCR product of 322 bp. In the presence of A3243G mutation, the PCR product was cleaved by ApaI to two fragments of 159 and 163 bp (14). The ApaI-digested PCR products were separated on a 3% agarose gel. Free software, Scion Image for Windows (Beta 4.02;http://www.scioncorp.com), was used to analyze the ratio of the signal intensity for the 159/163-bp band to the sum of the intensities for the 322-bp and 159/163-bp bands. RFLP analysis was also performed by electrophoresis of HaeIII digest of the PCR products on 5% polyacrylamide gels and calculation of ratios by the same software (8).

	Results

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Thirty-three DNA samples showing positive A3243G mutant signals and 3 samples (samples 30–32 in Fig. 1 ) negative on ASO analysis (Fig. 1A ) were reanalyzed by RFLP–gel electrophoresis (Fig. 1B ). As shown in Fig. 1 , three samples with low proportions of mutant A3243G mtDNA readily detected by ASO were not detected by RFLP with EtBr staining (Fig. 1B , sample 17, 20, and 29). In addition, five samples were barely detected by RFLP with EtBr staining (Fig. 1B , samples 3, 7, 12, 28, and 34). However, ASO analysis detects only the presence of the mutation, it does not allow quantification of the degree of heteroplasmy. All samples were then reevaluated by RT ARMS-qPCR.

For RT ARMS-qPCR analysis, calibration curves for the wild-type A3243 and mutant 3243G sequences were constructed. There was excellent correlation between the cycle number and mtDNA copy number with correlation coefficients of 1.000 and 0.999 for the wild-type and mutant target sequences, respectively. To validate the method, we mixed known amounts of the wild-type and mutant mtDNA to obtain 13 samples containing various proportions of mutant mtDNA. Results of the RT ARMS-qPCR are shown in Fig. 2 . The correlation coefficient (R²) for the observed vs expected proportion of mutant was 0.9995. A proportion of mutant mtDNA in samples as low as 0.1% was detected by this method.

View larger version (20K): [in this window] [in a new window]   Figure 2. Correlation of expected and observed percentages of A3243G mutant mtDNA by RT ARMS-qPCR analysis of 13 samples containing various proportions of mutant mtDNA. Equation for the line: y = 1.0042x + 0.26% (R2 = 0.9995).

To assess the potential usefulness of this method for the detection of unknown mutant A3243G heteroplasmy, we reevaluated the samples that had been analyzed by both the ASO and RFLP methods. We obtained mutant heteroplasmy proportions of 0.5%, 3%, and 4% for samples 29, 17, and 20, respectively, which had not been detected by RFLP with EtBr staining. For samples 30–32, which were negative by both radioactive ASO and RFLP–EtBr analysis, the qPCR analysis gave a value of 0.03%, close to the mean (SD) value of 0.03 (0.003)% for 12 wild-type controls. When we compared the results obtained by qPCR analysis with the results obtained by two independent RFLP analyses (Fig. 3 ), the correlation coefficient was 0.935.

View larger version (16K): [in this window] [in a new window]   Figure 3. Correlation of proportions of mutant A3243G mtDNA obtained by RFLP and qPCR. The RFLP values were the means (SD; error bars) of two independent measurements from agarose gel analysis of ApaI digest and polyacrylamide gel analysis of HaeIII digest. Equation for the line: y = 1.1x + 2.6% (R2 = 0.9348).

	Discussion

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There are several disadvantages of using conventional RFLP analysis in the quantification of mutant mtDNA heteroplasmy. One disadvantage is that, compared with radioactive methods, the detection sensitivity of EtBr is low. It is usually difficult to measure mutant heteroplasmy <10%. Another disadvantage is that RFLP analysis can be complicated by incomplete digestion with the restriction enzyme, which leads to underestimation of the percentage of heteroplasmy. In addition, nonspecific PCR products may interfere with the analysis. More commonly, the last-cycle hot PCR-RFLP method is used for quantification of the degree of mutant heteroplasmy to eliminate inaccuracies introduced by the formation of heteroduplex (15). However, this method involves several steps, including PCR, radioactive labeling, restriction enzyme digestion of hot material, gel electrophoresis, and analysis of the signal intensity. Furthermore, the accuracy is greatly reduced if the DNA sample is overloaded or the film is overexposed. RT ARMS-qPCR analysis overcomes these problems. In this method, PCR, signal recording, and analysis are all done in one step. When the PCR is completed, the data also have been collected and analyzed. Thus, RT ARMS-qPCR is a sensitive, nonradioactive method that can detect and quantify heteroplasmic mtDNA mutations in a one-step reaction. The ASO method, although sensitive and capable of being multiplexed, does not allow quantification of heteroplasmies. Quantification of mutant mtDNA heteroplasmies is important to confirm the carrier status of patients for mtDNA disorders.

Use of RT ARMS-qPCR for the measurement of mutant mtDNA heteroplasmy has several advantages. Generally, target sequences are amplified, and sequence-specific TaqMan (16) or Molecular Beacon (17) probes for the mutant or wild type are used for detection and quantification. These methods require the synthesis of each specific labeled probe, which can be costly. Our approach is to design target-sequence-specific ARMS primers for specific amplification of the target sequence followed by SYBR Green binding and reporting of the PCR products. It therefore eliminates the need to synthesize expensive fluorescent probes and can be easily expanded for the analysis of other common mtDNA mutations, such as A8344G for the MERRF (myoclonic epilepsy and ragged red fiber) syndrome, T8993G and T8993C for NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome, and the detection of homoplasmic LHON (Leber hereditary optic neuropathy) mutations. It not only detects and quantifies the heteroplasmic mtDNA mutation in a single step, but also makes it possible to determine the total amount of mtDNA genome if a standard nuclear gene target sequence is included in the analysis for normalization (17). This is particularly useful if mitochondrial proliferation is being considered, as in the case of the A8344G mutation (17). The method could allow simultaneous quantification of total mtDNA genome content in addition to detection and quantification of point mutations. Using this method, we have recently demonstrated the compensatory amplification of mtDNA in a mildly affected patient who harbored 92% deletion mutant mtDNA (18). RT-qPCR has been widely used to monitor mtDNA depletion in HIV-infected patients receiving prolonged nucleoside analog reverse transcriptase inhibitor therapy (10)(19). It has also been used to quantify mtDNA deletions (18)(20). We believe that RT ARMS-qPCR could ultimately provide a one-step technique for both qualitative and quantitative molecular analysis of mtDNA defects.

In conclusion, we present a rapid, sensitive, and reliable one-step RT-qPCR method for quantification of mutant mtDNA heteroplasmies. This method could be used for the quantification of heteroplasmy for any given mtDNA point mutation.

	Acknowledgments

 
This study was supported in part by a grant from the Muscular Dystrophy Association (to L.J. Wong). We thank Dr. Li Guo for technical assistance in ASO analyses.

	Footnotes

 
¹ Nonstandard abbreviations: mtDNA, mitochondrial DNA; RFLP, restriction fragment length polymorphism; EtBr, ethidium bromide; ASO, allele-specific oligonucleotide; RT-qPCR, real-time quantitative PCR; ARMS, amplification refractory mutation system; and C_T, threshold cycle.

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: clinchem.2004.031153v1 50/6/996    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (39) Citing Articles via Google Scholar Google Scholar Articles by Bai, R.-K. Articles by Wong, L.-J. C. Search for Related Content PubMed PubMed Citation Articles by Bai, R.-K. Articles by Wong, L.-J. C. Related Collections Molecular Diagnostics and Genetics