| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Molecular Diagnostics and Genetics |
1 Department of Clinical Chemistry and Laboratory Medicine, Kyushu University, Graduate School of Medical Sciences, Fukuoka, Japan.
2 Department of Biology Education, Daegue University, Kyungsan, Korea.
3 Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Nakornpathom, Thailand.
aAddress correspondence to this author at: Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan. Fax 81-92-642-5772; e-mail kang{at}mailserver.med.kyushu-u.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Methods: Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3' end matched to nucleotide position 3243 of the mutant.
Results: With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5–100 ng; annealing temperature, 61–66 °C; and PNA, 1.5–3.5 µmol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed <0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant.
Conclusions: PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Mitochondrial mutations are broadly classified into two groups: rearrangements (deletions and duplications) and point mutations. Among point mutations, an A-to-G mutation at nucleotide position (np) 3243 in the human mitochondrial tRNALeu(UUR) gene (A3243G) is the most common. This particular mutation accounts for 
80% of patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS)(2)(3). The percentage of cells containing mtDNA with the heteroplasmic A3243G mutation varies from tissue to tissue as described above and may be highest in affected tissues such as muscle and brain. However, samples that may be easily attained in a noninvasive manner and routinely used, such as blood or urinary cells, usually show lower percentages of heteroplasmy. For example, the A3243G mutation was detected in the blood of only 5 of 10 patients but was detected in the muscle of all 10 patients(4).
In addition to the classic mitochondrial encephalomyopathies such as MELAS, the A3243G mtDNA mutation has been shown to be involved type II diabetes mellitus (DM) and aging(5). Normal ATP production in mitochondria is critical, particularly for insulin secretion from pancreatic beta cells(6). Consistent with this fact, individuals with various types of MELAS often have symptoms of diabetes(7)(8), and accumulation of the mutation in pancreatic beta cells could cause adult-onset DM. In fact, the mutation is also found in patients with DM who were not previously diagnosed with MELAS(9). Although many of these patients exhibit a variety of neurologic disorders, typically including deafness, the A3243G mutation has also been found in DM patients with few neuromuscular symptoms. Considering that the heteroplasmy may be highest in affected tissues, the pancreas may be a good source for examination of the A3243G mutation in patients with diabetes(10); however, pancreatic biopsy is not available for routine screening. Instead, peripheral leukocytes and urinary epithelial cells, which are obtained in a noninvasive manner, are commonly used in screening. The percentage of mtDNA mutations is usually higher in the latter than in the former(11)(12)(13).
The A3243G mutation creates a new restriction site for the restriction enzyme ApaI; thus, this mutation is typically surveyed by a conventional PCR-restriction fragment length polymorphism (RFLP) method in which a region including np 3243 is PCR-amplified, digested with ApaI, and then stained with ethidium bromide after agarose gel electrophoresis. This method can barely detect the heteroplasmy at concentrations of 5–10%(14). Although the prevalence of DM patients with the A3243G mutation is estimated to be 1–2% of all DM patients(8), it is highly likely that the A3243G mutation will be missed in some DM patients by the RFLP method using peripheral blood cells(14)(15). To address this issue, we previously developed a sensitive ligation-mediated PCR-based (LMPCR) method that is able to detect the A3243G heteroplasmy present at a concentration of 0.01%(16). With the LMPCR method, we were able to detect the heteroplasmy present at 0.01–0.1% in approximately one-half of 136 apparently healthy volunteers; no volunteer had more than 0.1%. On the other hand, we found that the heteroplasmy was present in concentrations >0.1% in the leukocytes of 1% of 233 patients with type II DM.
This LMPCR method is very specific and highly sensitive, but it is only semiquantitative, in addition to being somewhat laborious and time-consuming. Thus, it is not an ideal routine clinical test, especially for large numbers of samples. We developed a sensitive quantitative method that combines peptide nucleic acid (PNA) and allele-specific PCR (Fig. 1
, bottom panel). This combination increases the detection of the A3243 heteroplasmy by approximately two orders of magnitude more than use of PNA-directed PCR-clamping alone (Fig. 1
, top panel).
|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
preparation of dna
The total DNA of the cell lines and peripheral leukocytes was extracted with QIAamp DNA extraction reagents (QIAGEN). The DNA was treated with RNase A, extracted with phenol–chloroform (1:1 by volume), precipitated with ethanol, resolubilized in 20 µL of distilled water, and quantified based on the absorbance at 260 nm.
pna-assisted allele-specific pcr
PNA (5'-ACCGGGCTCTGCCAT-3'), which was designed to bind the L-strand, was obtained from FASMAC Co., Ltd. A sense primer, mtL1-1 (5'-CAT AAC ACA GCA AGA CGA GAA GAC CCT ATG G-3'), and an antisense primer, mt3243HC (5'-TTT TAT GCG ATT ACC GGG CC-3'), were used. The standard PCR reaction mixture consisted of 1x LightCycler mixture (LC-FastStart Reaction Mix SYBR GREEN I; Roche) containing the DNA-binding fluorescent dye SYBR Green I and Taq DNA polymerase, 2.5 µM PNA, 0.25 µM each primer, and 10 ng of total DNA in 20 µL. Thermal cycling was conducted in a LightCycler. The standard conditions were as follows: an initial DNA denaturation step of 10 min at 94 °C and an amplification step of 20 s at 94 °C, 5 s at 77 °C, 5 s at 70 °C, 10 s at 64 °C, and 20 s at 72 °C. The 5 s at 77 °C and 5 s at 70 °C steps were inserted to slow the temperature decrease and allow binding of PNA to DNA. DNA amplification was monitored in real time.
quantification of MTDNA
An 300-bp DNA fragment (np 16052–16361) was PCR-amplified with sense primer 5mt16052 (5'-CCA CCC AAG TAT TGA CTC ACC C-3') and antisense primer 3mt339 (5'-CGA GAA GGG ATT TGA CTG TAA TG-3'). The PCR product was cloned into a TA vector, pQTmt. The plasmid was quantified by the absorbance at 260 nm and used as the calibrator for total mtDNA (wild-type and the A3243G mutant) quantification. The PCR reaction mixture for the quantification consisted of 1x LightCycler mixture, 2.0 mM MgCl2, 0.5 µM each primer, and various amounts of the pQTmt plasmid in 20 µL. Thermal cycling was conducted in a LightCycler. The standard thermocycling conditions were as follows: an initial DNA denaturation step of 10 min at 95 °C and an amplification step of 15 s at 95 °C, 5 s at 60 °C, and 15 s at 72 °C.
Similarly, an 560-bp DNA fragment including the A3243G mutation (np 2703–3262) was PCR-amplified with mtL1-1 and antisense primer mt3243HC (the same as for the allele-specific PCR) and cloned into a TA vector; we named this plasmid pQMmt and used it as the calibrator for the A3243G mutant.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
). When we performed allele-specific PCR in the presence of PNA using 10 ng of total DNA in 20 µL of the reaction mixture, the crossing point of 2SD was 15 cycles earlier than that of 2SA (Fig. 2A
). The final fluorescent intensity of SYBR Green I, which indicates the amount of double-stranded DNA, was 30% lower in 2SA than in 2SD. We mixed the two total DNAs at various ratios (from 0.1% to 10% 2SD) and found that 2SA DNA with 0.1% 2SD added could be clearly distinguished from 100% 2SA (Fig. 2A
). By constructing a calibration curve of crossing points vs percentage of 2SD (100% to 0.1%; Fig. 2B
), we could estimate a 2SA concentration that corresponded to a mean (SD) of 0.014 (0.0008)% of 2SD (n = 3). Given that 2SA is 100% wild type, the amplification of wild-type mtDNA was apparently suppressed 7000-fold compared with the A3243G mutant in this PNA-assisted allele-specific PCR. The PCR products at the endpoint of the reaction (i.e., after 50 cycles) are shown in Fig. 2C
.
|
|
The PCR products for 2SD were found at their anticipated band length of 560 bp (Fig. 2C
, lane 1). However, in the case of 2SA, the 560-bp product was much less prominent, and a lower band that may be derived from the presence of "primer-dimers" was detected (Fig. 2C
, lane 5). The lower band was also observed in the absence of mtDNA (Fig. 2C
, lane 6). Notably, the lower primer-dimer band was hardly seen even in 0.1% 2SD (Fig. 2C
, lane 4), suggesting that the primer-dimer is formed only when the amount of the mutant 3243 mtDNA is extremely low or absent. From these results, the erroneous amplification of wild-type mtDNA may actually be much lower than that estimated by the fluorescence. As shown in the melting curves of the PCR products (Fig. 2D
), the 560-bp product and the primer-dimer were readily distinguished, with the former having a higher melting temperature (Tm) than the latter (Fig. 2D
).
We also examined whether this 15-cycle difference is maintained at different concentrations of total DNA. We varied the DNA amount from 1 to 100 ng. The 15-cycle difference was maintained between 5 and 100 ng (see Table 1
in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol50/issue11/). In addition, we changed the annealing temperatures from 60 °C to 68 °C. The 15-cycle difference was not affected much between 61 and 66 °C (Table 2 in the online Data Supplement). It is striking that the allele-specific PCR allows such a broad range of annealing temperatures because usually strict control of annealing temperatures is critical for assuring high specificity, particularly in cases with heteroplasmy <1%. Finally, we examined the effect of the PNA concentration. Without PNA, allele-specific PCR revealed an 10-cycle difference between 2SA and 2SD, whereas the presence of PNA increased the difference by 5 cycles (Table 3 in the online Data Supplement). These results suggest that allele-specific PCR plus PNA increases the specificity of amplification of the A3243G mutant by two orders of magnitude more than does PNA alone. The 15-cycle difference was observed with PNA concentrations between 1.5 and 3.5 µmol/L (Table 3 in the online Data Supplement).
determination of MTDNA copy number
To quantitatively estimate the heteroplasmy, we determined the copy numbers of the mutant and total mtDNA. For the former, plasmid pQMmt, which includes a DNA fragment (np 2703–3262) containing the A3243G mutation was constructed and used as the copy number calibrator for the mutant mtDNA. We also added PNA in the PCR reaction mixtures for the calibration curve to adjust the amplification efficiency of the mutant mtDNA. For the latter, plasmid pQTmt, which includes a DNA fragment (np 16052–16361), was constructed and used as the calibrator for determining copy number. Using these two plasmids, we measured the copy numbers of mutant and total mtDNA in 2SA and 2SD cells and then calculated the heteroplasmy. The heteroplasmy of 2SD cells was 97.3%, which is close to 100% (Table 1
). The value for 2SA (0.013%) is consistent with the estimate that was obtained by mixing 2SA and 2SD (Fig. 2
) and suggests that 0.02% heteroplasmy is a background in this measurement system. The mixture of 2SA and 2SD DNA exhibited amounts of heteroplasmy close to the mixing ratio (Table 1
).
We next measured the heteroplasmy in blood samples from 40 apparently healthy volunteers. When examining the specimens from the volunteers, we always measured 2SD in parallel. The obtained heteroplasmy value of the 2SD sample was divided by a factor and adjusted to 100%. For example, when the assay found 95% heteroplasmy for 2SD from the measured copy numbers of mutant and wild-type mtDNA, we divided the 95% by a factor of 0.95, and the same factor was applied to the values for the specimens to minimize measurement errors. When we used these techniques, no volunteer had more than 0.06% heteroplasmy [mean (SD), 0.03 (0.01)%]. We therefore concluded that finding 0.1% heteroplasmy is a reliable indication of the presence of the mutation.
We then tested this method with two MELAS patients, P1 and P2 (80% and 20% heteroplasmy, respectively, according to our method; Fig. 3A
). The results of conventional PCR-RFLP analysis of those samples are shown in Fig. 3B
. The results for patient P2 were markedly different from those for 2SA in the PNA-assisted allele-specific PCR assay (Fig. 3A
), but the ApaI-cleaved bands in the sample from patient P2 were only weakly visible in the PCR-RFLP gel (Fig. 3B
). The DNA from patient P1 was diluted 8-, 80-, and 800-fold with DNA of a healthy individual to make 10%, 1%, and 0.1% heteroplasmy, and the resulting theoretical 0.1% heteroplasmy was clearly distinguished from the DNA of a healthy volunteer (Fig. 3C
). These results suggest that this new method is similarly sensitive for DNA extracted from peripheral blood cells from MELAS patients. We also examined 50 patients with type II DM and found 2 positive individuals, with 20% and 40% heteroplasmy, respectively (results not shown).
|
Murdock et al.(18) have reported that 0.1% heteroplasmy of the A3243G mutation is detected by a PNA-directed PCR-clamping method. However, in their report, the PCR product for the mutation was barely detectable at a 1% concentration when wild-type and mutant DNA were mixed at 100:1. Thus, they needed to perform a second round of PCR to make the signals visible. In addition, digestion of the second-round PCR products with a restriction enzyme was required to confirm the presence of the mutation because wild-type mtDNA was significantly amplified even at 1% in their system. Hancock et al.(19) also reported detection of 0.1% heteroplasmy by PNA-directed PCR clamping. Similarly, they needed to sequence the PCR products to confirm the A3243G mutation and to ensure that no other mutation interfered with the PNA binding to the wild type. Initially, we also attempted simple PNA-directed PCR clamping under many conditions, but we never succeeded in completely selectively amplifying the mutant mtDNA, even at 1%; i.e., wild-type mtDNA was always significantly amplified. Thus, under the present conditions, it seems to be impossible to completely suppress the amplification of wild-type mtDNA with PNA alone when the heteroplasmy is 1% and the wild type is 99%. An advantage to using a primer that does not overlap the 3243 site (Fig. 1
, top panel) is that amplification of the mutant mtDNA can be definitively confirmed by enzyme digestion or sequencing(18)(19). However, in our PNA-assisted allele-specific PCR (Fig. 1
, bottom panel), we did not take this approach because we wished to develop a simple but quantitative method that can be performed in a typical clinical laboratory. Our method provides high specificity over a broad range of PCR conditions. Especially striking is that selective amplification is maintained over a wide range of annealing temperatures. This is particularly important from the point of view of routine clinical tests because we frequently encounter the problem of undesirable amplification by allele-specific PCR as a result of small fluctuations in the annealing temperature in PCR instruments, particularly when we are trying to detect very low percentages of heteroplasmy. We believe that the simple addition of PNA to any allele-specific PCR may generally improve the stability of allele-specific amplification.
The Tm of PNA/DNA is usually reduced by one base mismatch much more than is that of wild-type DNA/DNA. The PNA-directed PCR clamping is strongly based on this principle. However, this strong dependence on base matching could create an adverse situation if there is single-nucleotide polymorphism (SNP) in the region of PNA clamping, such that the wild-type genome is falsely amplified. This is also true for our PNA-assisted allele-specific PCR, although it is not totally dependent on the Tm of PNA. Fortunately, we did not find any SNPs from np 3236 to np 3250, the clamping region of our PNA, in the databases of Ingman et al. (76 persons)(20), Finnila et al. (192 persons)(21), Kong et al. (48 persons)(22), or Herrnstadt et al. (560 persons)(23). No SNPs in this region are found in the Japanese database for 1000 Japanese(24) or in the US database(1). Thus, this region is well conserved, and SNPs in the region may be extremely rare. Two cases with Kearns–Sayre syndrome are reported to harbor mutations at 3249 and 3250, respectively(1). For these cases, a false positive may be better than a false negative. A patient who has a false-positive result for A3243G would then receive further examination for confirmation.
In conclusion, we show that 0.1% heteroplasmy is reliably and quantitatively detected by PNA-assisted allele-specific PCR and that the value 0.1% is not simply the lower limit of detection. When we previously measured heteroplasmy by LMPCR, we detected 0.01% heteroplasmy in peripheral blood cells from approximately one half of the healthy individuals and DM patients, but no healthy individuals had >0.1% heteroplasmy(16). This observation was confirmed by the method presented in this report. Murdock et al.(18) also reported that the A3243G mutation does not typically accumulate above 0.1% with age, even in muscle or brain. Therefore, the presence of heteroplasmy >0.1% may be diagnostically significant. It is well established that healthy people typically harbor very low concentrations of the A3243G heteroplasmy(18)(25). Recently, the authors of a case study reported that the concentration of the 3243 mutation in peripheral blood cells of a patient with mitochondrial diabetes was 0.102%(26). Thus, the quantitative detection of 0.1% heteroplasmy appears to be a required and sufficient prerequisite for a practical clinical test for the A3243G mutation. Our method feasibly satisfies this condition and thus may be suitable for a routine clinical test. Because collecting urine samples is less invasive than collecting blood and the concentration of mtDNA mutations is usually much higher in urine samples than in blood samples(11)(12)(13), application of this method to urine samples may be even more useful as a routine clinical test.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  K. S. Lim, R. K. Naviaux, and R. H. Haas Quantitative Mitochondrial DNA Mutation Analysis by Denaturing HPLC Clin. Chem., June 1, 2007; 53(6): 1046 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Singh, S. Ellard, A. Hattersley, and L. W. Harries Rapid and Sensitive Real-Time Polymerase Chain Reaction Method for Detection and Quantification of 3243A>G Mitochondrial Point Mutation J. Mol. Diagn., May 1, 2006; 8(2): 225 - 230. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
