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Direct detection of multiple point mutations in mitochondrial DNA

^a Author for correspondence. Fax 213-666-0489; e-mail lcwong{at}hsc.usc.edu

	Abstract

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Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA^leu(UUR), tRNA^lys/ATPase, and ND₄ regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
Mitochondria are eukaryotic cytoplasmic organelles where oxidative phosphorylation takes place. Electrons pass from NADH and FADH₂ to complexes I through IV in the mitochondrial inner membrane. The proton gradient generated by the stepwise transfer of electrons is used to phosphorylate ADP to form ATP by complex V, the ATP synthetase. The circular 16.6-kb human mitochondrial genome has been completely sequenced. It encodes 13 protein subunits of the complexes in the oxidative phosphorylation pathway, the 12S and 16S rRNA, and 22 tRNAs required for mitochondrial protein synthesis. Importantly, mitochondrial biogenesis and maintenance also involves nuclear DNA (nDNA)-encoded proteins such that all essential mitochondrial processes such as replication, transcription, and translation require nDNA-encoded factors (1)(2).¹ Therefore, genetic defects of mitochondrial function can be caused by mutations in either nuclear or mitochondrial genes.

There are numerous mitochondria in each cell, usually hundreds to thousands, each having 2–10 copies of mitochondrial DNA (mtDNA) (3). Because virtually all the mtDNA of a fertilized egg are derived from the oocyte, maternal inheritance of mtDNA defines the mode of transmission of mtDNA disorders. A mutation in mtDNA may be present in all mtDNA copies (homoplasmy) or coexist with normal mtDNA copies (heteroplasmy). Usually, benign polymorphisms of mtDNA are homoplasmic and pathogenic mutations are heteroplasmic. The variable phenotypic expression of pathogenic mutations depends upon the degree of heteroplasmy and the energy requirements of the affected tissue. Accordingly, each tissue has a threshold concentration of mutant mtDNA that must be exceeded to cause respiratory chain dysfunction (4). This threshold concentration also varies among different mtDNA point mutations.

Because mitochondrial disorders affect a variety of organ systems, a diverse group of systemic diseases have been associated with mitochondrial mutations. Neurological manifestations of mitochondrial diseases include ophthalmoplegia, seizures, sensorineural hearing loss, myoclonus, optic neuropathy, pigmentary retinopathy, myopathy, ataxia, dementia, and peripheral neuropathy (3). Nonneurological systemic manifestations include cardiac conduction defects, cardiomyopathy, diabetes mellitus, hypoparathyroidism, cataracts, lactic acidosis, intestinal pseudoobstruction, and pancreatic dysfunction (3). Both large deletions and point mutations in mtDNA have been identified in patients with characteristic oxidative phosphorylation defects. The most common point mutations are A3243G, accounting for 80% of patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); A8344G, which underlies myoclonic epilepsy, ragged red fibers (MERRF); T8993G/C, leading to neuropathy, ataxia, retinitis pigmentosa (NARP); and G11778A, found in >50% of patients with Leber hereditary optic neuropathy (LHON). Genetic heterogeneity exists whereby different mutations may result in similar disease presentation. For example, A3243G, T3271C, T3291C, and C3256T can all present as a MELAS-like phenotype. Correspondingly, mitochondrial disease is also characterized by phenotypic pleiotropy whereby the identical point mutation may lead to different disease phenotypes. An example is the A3243G MELAS mutation, also found in some patients with maternally inherited diabetes/deafness (5).

To date, the mainstay of mitochondrial point mutation analysis has been PCR amplification of the mtDNA region of interest, followed by restriction enzyme analysis for each known mutation. However, given the complexity and heterogeneity of the mtDNA disorders, analysis of multiple point mutations is often desirable. In many cases, at the time when molecular diagnosis is requested, the clinical phenotype is nonspecific or evolving. Nevertheless, a definitive molecular diagnosis to rule out common mutations in mtDNA is necessary for effective patient management and genetic counseling. Analysis of several mutations by single PCR/restriction enzyme fragment length polymorphism (RFLP) is not practical. Here we describe a simple, sensitive, accurate, and cost-effective multiplex PCR/allele-specific oligonucleotide (ASO) method for the simultaneous screening of multiple point mutations in mtDNA.

	Patients and Methods

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Samples from patients suspected of mtDNA disorders were submitted to the Molecular Genetics Laboratory at Children's Hospital Los Angeles for mutation analysis.

Genomic DNA was extracted from peripheral blood, muscle, hair follicles, cheek cells, or paraffin-embedded tissues with published procedures (6)(7)(8). Three mutation hot spots, including tRNA^leu(UUR), tRNA^lys/ATPase, and ND₄ regions, were PCR-amplified with the three pairs of primers listed in Table 1 . Ninety-six-well plates were used for multiplex PCR amplification with Perkin-Elmer's 9600 thermal cycler. Each 100 µL of PCR mixture contained 1x Promega PCR buffer; 1.5 mmol/L MgCl₂; 0.2 mmol/L of each dNTP; 0.5 µmol/L each of primers mt1, mt12, mt3, mt8, LHON-F, and mt10; 1 U of Taq DNA polymerase; and 100 ng of genomic DNA. The reaction mixture was denatured at 94 °C for 5 min, followed by 25 cycles of 45 s of denaturation at 94 °C, 1 min of reannealing at 55 °C, and 2 min of extension at 72 °C. The PCR is completed by a final extension cycle at 72 °C for 5 min. Two microliters of PCR product was spotted onto a Biodyne B+ membrane. Dot blots were prepared for each of the following normal and mutant probes: A3243, A8344, G11778, A3243G, T3271C, A8344G, T8356C, T8993C, T8993G, G8363A, G3460A, T3394C, and G11778A. The normal probes are used to ensure that each region has been PCR-amplified. Eight rare point mutations were studied by hot pools I and II. Hot pool I contained a mixture of mutant probes A3251G, A3252G, A3260G, and A3302G. Hot pool II contained a mixture of mutant probes T3250C, C3256T, T3291C, and C3303T. The sequences of the ASO probes for various mutations are listed in Table 2 . Membrane preparation and hybridization conditions were those of published procedures (9). The ASO probes were end labeled with [-³²P]ATP with polynucleotide kinase. Each dot blot was placed in a 15- or 50-mL disposable tube to be hybridized with specific probe. Multiple tubes were put in a hybridization bottle. The wash temperature varied with the melting temperature (T_m) of each ASO probe and was determined experimentally (Table 2 ). The proportion of mutant mtDNA was quantitatively analyzed according to published procedures (7)(10)(11).

	Results

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Approximately 2000 specimens submitted to the Molecular Genetics Laboratory at Children's Hospital Los Angeles for molecular diagnosis of mtDNA disorders were analyzed by the multiplex PCR/ASO hybridization procedures described above. Representative hybridization blots are illustrated in Figs. 1–3 . Positive hybridization signals with mutant probes indicate the presence of the mutant mtDNA. The variation in signal intensities within the same blot is secondary to the presence of different proportions of mutant mtDNA, heteroplasmy, which can be further quantified by last-cycle hot PCR/RFLP and measurement of the radioactivity in normal and mutant DNA bands (7)(10)(11). The most common mtDNA point mutation found among the samples we analyzed is the A3243G MELAS mutation (Fig. 1 , panel C), followed by the T8993G/C NARP mutation (Fig. 2 , panels B and C). Additional mutations detected in at least one family include T3271C for MELAS (Fig. 1 , panel B); A8344G (Fig. 2 , panel D) and T8356C (Fig. 1 , panel D) for MERRF; G11778A, G3460A (Fig. 3 , panel C), and T3394C (Fig. 3 , panel D) for LHON; and G8363A (Fig. 3 , panel B) for cardiomyopathy. Other rare mutations such as T3250C, A3251G, A3252G, C3256T, A3260G, T3291C, A3302G, and C3303T that account for myopathy, cardiomyopathy, and multisystem disorders were also included in this study. Only one family with A3302G mutation was detected. Quantitative analysis (11)(12) was performed on specimens that showed positive signals for A3243G to evaluate the sensitivity of this method in the detection of mutant heteroplasmy. Fig. 4 illustrates that a proportion of mutant mtDNA as low as 2.5% was readily detectable. Over 1000 samples were analyzed with both the traditional PCR/RFLP method (results not shown) and the multiplex PCR/ASO method. The results demonstrated that the PCR/ASO method is more sensitive. This is on the basis that two mutations, a 6% A3243G mutation and a 17% T8993C mutation, were not detected by PCR/RFLP method but were readily detected by the multiplex PCR/ASO method. Reexamination of the PCR/RFLP data indicated that the mutant bands of T8993C mutation were very faint.

View larger version (94K): [in this window] [in a new window]   Figure 1. ASO dot-blot analysis of point mutations in mtDNA. The probes used for hybridization in panels A, B, C, and D are normal A3243, mutant T3271C, mutant A3243G, and mutant T8356C, respectively. The A1 position is either the normal or the positive mutant control.

View larger version (94K): [in this window] [in a new window]   Figure 2. ASO dot-blot analysis of point mutations in mtDNA. The probes used for hybridization in panels A, B, C, and D are normal T8993, mutant T8993G, mutant T8993C, and mutant A8344G, respectively. The A1 position is either the normal or the positive mutant control.

View larger version (89K): [in this window] [in a new window]   Figure 3. ASO dot-blot analysis of point mutations in mtDNA. The probes used for hybridization in panels A, B, C, and D are normal G11778, mutant G8363A, mutant G11778A plus G3460A hot pool, and mutant T3394C, respectively. The A1 position is either the normal or the positive mutant control.

View larger version (32K): [in this window] [in a new window]   Figure 4. ASO analysis of MELAS with various percentages of A3243G mutant mtDNA. The percentage of mutant DNA was determined by last-cycle hot PCR/RFLP method (11)(12).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The ASO dot-blot method has been widely applied to the detection of known point mutations underlying several genetic diseases. DeMarchi et al. described a robotic method for the analysis of 23 point mutations in the CFTR gene (9). This method is much less expensive than RFLP analysis, especially when a large number of specimens and multiple point mutations are being analyzed. For nuclear genes, the ASO analysis of heterozygotes gives signals with both mutant and normal probes. The signal intensities are usually quite uniform. However, the heteroplasmy of mtDNA mutations from 0% to 100% yields variable signal intensities with ASO analysis. The sensitivity of detection by ASO method as shown in Fig. 2 is 2.5%. If positive signals are detected on an ASO blot, quantitative analysis of the radioactive-labeled PCR products by RFLP is performed to determine the percentage of mutant mtDNA in the specimen (12). Traditional methods involving agarose gel/RFLP analysis by ethidium bromide detection is limited in that a mutant heteroplasmy of low amount may not be readily detected. When the distinction between mutant and normal DNA is based on the gain or loss of a restriction site, incomplete digestion with the restriction enzyme can lead to an inaccurate result. The intensities of the mutant bands also depend on the size of the DNA fragment. In fact, by using the multiplex PCR/ASO method, we were able to detect a 6% A3243G and a 17% T8993C heteroplasmy that were missed in the earlier diagnosis with the traditional PCR/RFLP/agarose gel method. Furthermore, not all mutations will result in gain or loss of a restriction site. In such cases, modified primers are needed for PCR to create a restriction site. This procedure will increase the cost of the DNA testing and may also cause problems in PCR amplification. Finally, it is easier and less expensive to make multiple probes for hybridization than to operate multiple PCRs, possibly each under different conditions, and to perform multiple RFLP agarose gel analysis. An alternative method is the reverse dot-blot analysis. A study of five homoplasmic LHON point mutations using the reverse dot method was recently reported (13). However, in our experience, the pathogenic mutations in children are usually heteroplasmic, and may not be detectable by this method if the proportion of mutant mtDNA is low. It would also be very difficult to optimize a single condition for 18 or more probes.

Genetic heterogeneity complicates mitochondrial genotype/phenotype correlation. For instance, at least four different point mutations present as a MELAS-like syndrome, and at least 10 different point mutations have been found in patients with LHON. The A3243G mutation can have several different clinical manifestations, such as MELAS (14), diabetes and hearing loss with or without macular-pattern retinal dystrophy (5)(15)(16), and chronic progressive external ophthalmoplegia. The histologic and biochemical phenotype can be nonspecific. The clinical phenotype of patients with A3243G overlaps with that of patients with A8344G in that they may both have ragged red fibers and lactic acidosis. Thus, the mitochondrial disorders are a complex and heterogeneous group of genetic disorders. To rule out the most common point mutations in mtDNA, several point mutations causing MELAS, MERRF, NARP, Leigh, and (or) LHON syndromes may need to be evaluated. The individual analysis of multiple point mutations is time consuming, labor intensive, expensive, and not practical. The multiplex PCR/ASO method described here is direct, simple, accurate, sensitive, and cost effective. Accordingly, our turnaround time for the analysis of 18 point mutations is ~3–4 days. Complete molecular screening of mutations in mtDNA should also include Southern transfer analysis to detect large DNA deletions.

Because multiplex PCR amplifies the regions containing mutation hot spots, as new mutations in these regions are identified, the same blots can be stripped and reanalyzed with the new probes. This would permit efficient analysis of several specimens for the presence of the newly identified mutations. Currently, we have improved the multiplex PCR/ASO method to analyze 45 known point mutations in the mtDNA genome. The PCR products of mtDNA from all the patients are in the blots that can be easily reused in the future for the detection of any newly discovered mutations in these regions.

Molecular diagnosis is only part of a complete workup of mitochondrial disease. Patients highly suspected of mtDNA disorders on the basis of clinical, pedigree, and (or) laboratory information should receive a comprehensive evaluation including histochemical/electron microscopic studies, enzyme assays of electron transport chain, and mtDNA mutation analysis. In our experience, only ~7% of the specimens sent to our laboratory for DNA testing will have detectable pathogenic mutations by multiplex PCR/ASO and Southern analysis. Possibilities for further molecular characterization include single-strand conformation polymorphism analysis and direct sequencing of the entire mtDNA genome.

In the past, there has been uncertainty about the appropriate tissues to be tested for mtDNA mutations. Approximately 15% (300 of 2000) of the specimens tested are muscle tissue. Results of analysis of both blood and muscle specimens in 12 patients revealed that, in all cases, the blood contained a lower proportion of mutant mtDNA than muscle (17). Recently, we have looked at other noninvasive tissue specimens as alternatives. Among these are hair follicles and buccal mucosal cells. The results (17) indicate that although the percentage of mutant mtDNA fluctuates from tissue to tissue, in all patients analyzed, the mutant mtDNA for point mutations is consistently either present or absent in all three tissues (blood, hair follicles, and buccal cells) analyzed.

There is a bias in patient selection. Our laboratory is in a children's hospital. The majority of our patients have clinical presentations of MELAS, NARP, Leigh syndrome, and cardiomyopathy. mtDNA mutations that account for adult forms of myopathy, cardiomyopathy, MERRF, multisystem disorders, and mutations associated with aging such as Alzheimer and Parkinson diseases are apparently underrepresented.

	Acknowledgments

 
We thank Richard Boles and Dennis Johnson for critical review and many helpful discussions.

	Footnotes

 
Molecular Diagnostics Laboratory, MS #103, Children's Hospital Los Angeles, Los Angeles, CA 90027.

¹ Nonstandard abbreviations: nDNA, nuclear DNA; mtDNA, mitochondrial DNA; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy and ragged-red fibers; NARP, neuropathy, ataxia, retinitis pigmentosa; LHON, Leber hereditary optic neuropathy; RFLP, restriction fragment length polymorphism; and ASO, allele-specific oligonucleotide.

	References

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This Article Abstract Full Text (PDF) 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 ISI 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 ISI Web of Science (57) Citing Articles via Google Scholar Google Scholar Articles by Wong, L.-J. C. Articles by Senadheera, D. Search for Related Content PubMed PubMed Citation Articles by Wong, L.-J. C. Articles by Senadheera, D. Related Collections Molecular Diagnostics and Genetics Automation and Analytical Techniques