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Published in final edited form as: Mitochondrion. 2010 Jan 11;10(3):300–308. doi: 10.1016/j.mito.2010.01.003

Extensive screening system using suspension array technology to detect mitochondrial DNA point mutations

Yutaka Nishigaki a,*, Hitomi Ueno a, Jorida Coku b, Yasutoshi Koga c, Tatsuya Fujii d, Ko Sahashi e, Kazutoshi Nakano f, Makoto Yoneda g, Michiko Nonaka h, Linya Tang i, Chia-Wei Liou i, Veronique Paquis-Flucklinger j, Yasuo Harigaya k, Tohru Ibi e, Yu-ichi Goto l, Hiroko Hosoya a, Salvatore DiMauro b, Michio Hirano b, Masashi Tanaka a
PMCID: PMC7568344  NIHMSID: NIHMS1630799  PMID: 20064630

Abstract

We established an extensive and rapid system using suspension array to detect 61 representative mitochondrial DNA (mtDNA) heteroplasmic or homoplasmic point mutations (29 for Series A and 32 for Series B) in 22 genes: 1 each in MT-RNR1, -TV, -ND1, -TQ, -TW, -TC, and -TH genes; 2 each in MT-TN, -TG, -ND4, -TL2, -TE, and -CYB genes; 3 each in MT-ATP6, -ND3, and -ND5 genes; 4 each in MT-CO1 and -TK genes; 5 each in MT-TI, -TS1, and -ND6 genes; and 10 in the MT-TL1 gene. We carefully selected 5′-biotinylated primers and pooled primers for use in two sets of multiplex-PCR amplifications. To detect both mutant and wild-type mtDNA, even when polymorphisms were present near the target mutation sites, we designed specific oligonucleotide probes. By using the mtDNA point mutation detection system of Series A (29 mutations) and Series B (32 mutations), we screened a total of 3103 mutant sites in 107 DNA samples for Series A and 13,101 mutant sites in 397 DNA samples for Series B. We succeeded in determining 99.4% (Series A) and 99.6% (Series B) of the targeted mutant sites by use of the system. The 22 samples with the m.3243A>G heteroplasmic mutation revealed positive signals with both mutant- and wild-type-specific probes in this detection system with a detection limit of approximately 2%. This genetic screening platform is useful to reach a definitive diagnosis for mitochondrial diseases.

Keywords: mtDNA, Pathological mutation, Mitochondria, MELAS, Suspension array

1. Introduction

In humans, mitochondrial DNA (mtDNA) is a 16,569-bp (base pair) double-stranded circular molecule encoding 37 genes (Anderson et al., 1981; Andrews et al., 1999) and is exclusively inherited from mothers. Because the evolutionary rate of mtDNA is much higher than that of nuclear DNA (Pakendorf and Stoneking, 2005) and because mtDNA is highly polymorphic, we detected 2935 mitochondrial single nucleotide polymorphisms (mtSNPs) in 1419 East Asian individuals. On the basis of these mtDNA sequence data, we established an East Asia mtDNA phylogeny (Tanaka et al., 2004) to classify 110 mitochondrial subhaplogroups in a human mtSNP database (http://mtsnp.tmig.or.jp/mtsnp/index_e.shtml). Moreover, we developed a comprehensive mtSNP detection system using suspension array technology. Nowadays, suspension array analysis using multi-microspheres combined with multiple sequence-specific oligonucleotide (SSO) customized probes and flowmetry is in widespread use as a multiplex assay for large-scale screening applications (Dunbar, 2006). Using our suspension array-based mtSNP screening system, we have already analyzed mtSNPs in more than 4000 Japanese and Korean subjects (Fuku et al., 2007; Nishigaki et al., 2007a,b; Tanaka et al., 2007).

Because many mitochondrial diseases are caused by mtDNA point mutations, it is necessary to distinguish pathological mtDNA mutations from non-pathogenic mtSNPs, which are common. According to the mtDNA mutation database MITOMAP, (http://www.mitomap.org/ accessed on August 15, 2009), more than 200 mutations in mtDNA have been reported. Currently, to diagnose mitochondrial diseases at the molecular level, DNA from patients must typically undergo multiple sequencing tests. Recently, various new techniques have been developed to rapidly detect target single or multiple mtDNA mutations, e.g., denaturing high-performance liquid chromatography (DHPLC; Meierhofer et al., 2005; van Den Bosch et al., 2000), a detection system using a mismatch-specific DNA endonuclease (Bannwarth et al., 2008), SNaP shot technology using a multiplex method based on the dideoxy single-base extension of unlabeled oligonucleotide primers (Cassandrini et al., 2006), the Biplex Invader® assay by hybridization of two overlapping oligonucleotides to the target sequence (Mashima et al., 2004), the Pyrosequencing™ technology to detect and estimate heteroplasmic mtDNA point mutations (White et al., 2005), and the matrix-associated laser desorption/ionization time of flight (MALDI–TOF MS) assay (Elliott et al., 2008); and a complete mtDNA resequencing chip: Mitochip (Leveque et al., 2007; Maitra et al., 2004) has been developed. The Mitochip also utilizes hybridization for detecting mutations or polymorphisms. Although the version 2.0 of Mitochip has been improved for detecting mutations, this present version was not designed for analysis of Asian mtDNA; because it is based on the revised Cambridge reference sequence (Anderson et al., 1981; Andrews et al., 1999), which belongs to the European haplogroup H2.

In this paper, we described a simultaneous rapid screening system customized for the Japanese for detecting 61 representative mtDNA point mutations by use of the suspension array technology.

2. Materials and methods

2.1. Selection of mitochondrial DNA pathological mutations

To detect representative mtDNA pathological point mutations associated with major complications of mitochondrial diseases (e.g., myopathy, encephalopathy, diabetes mellitus, deafness, and cardiomyopathy), we carefully selected 61 mtDNA point mutations, as shown in Fig. 1 and Supplementary Tables 1 and 2. All the mutations are listed in the MITOMAP database.

Fig. 1.

Fig. 1.

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The mtDNA map of the 61 mtDNA pathological point mutations examined by the extensive suspension array-based detection system. The 29 mutations by Series A and 32 by Series B are shown in blue and green, respectively. The m.3243A>G heteroplasmic mutation in the MT-TL1 (tRNALeu(UUR)) gene causes several major mitochondrial disease phenotypes such as MELAS (Goto et al., 1990; Pavlakis et al., 1984), MIDD, chronic progressive external ophthalmoplegia (CPEO; Sotiriou et al., 2009), and mitochondrial diabetes. The m.8344A>G heteroplasmic mutation in the MT-TK (tRNALys) gene causes MERRF (Shoffner et al., 1990; Yoneda et al., 1990). The m.8993T>G and m.8993T>C heteroplasmic mutations in the MT-ATP6 gene cause MILS or NARP (de Vries et al., 1993; Holt et al., 1990). The m.1555A>G homoplasmic mutation in the MT-RNR1 gene causes non-syndromic sensorineural hearing loss (SNHL) and aminoglycoside-induced hearing loss (Prezant et al., 1993). The m.11778G>A in the MT-ND4 gene is the most common cause of LHON (Wallace et al., 1988). The m.14484T>C mutation in the MT-ND6 gene has been considered another pathogenic LHON mutation (Johns et al., 1992), and this mutation apparently tends to arise spontaneously on the haplogroup J background (Elliott et al., 2008). DCM, dilated cardiomyopathy; DEAF, maternally inherited deafness or aminoglycoside-induced deafness; DM, diabetes mellitus; DMDF, diabetes mellitus + deafness; EM, encephalomyopathy; ESOC, epilepsy, strokes, optic atrophy, and cognitive decline; FBSN, familial bilateral striatal necrosis; FICP, fatal infantile cardiomyopathy plus, a MELAS-associated cardiomyopathy; KSS, Kearns–Sayre’s syndrome; LDYT, Leber hereditary optic neuropathy and dystonia; MHCM, maternally inherited hypertrophic cardiomyopathy; MICM maternally inherited cardiomyopathy; MM, mitochondrial myopathy; MMC, maternal myopathy and cardiomyopathy; MS, multiple sclerosis; PEM, progressive encephalomyopathy. Abbreviations and information about mutations are annotated in the MITOMAP database.

2.2. Analysis of DNA from patients, cybrids, and control subjects

All 79 DNA samples from 62 patients including 26 with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) or maternally inherited diabetes and deafness (MIDD), six with Leber hereditary optic neuropathy (LHON), four with maternally inherited Leigh syndrome (MILS), four cybrids from one patient with myoclonic epilepsy with ragged-red fibers (MERRF) and one with neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) were obtained from participating hospitals and institutions. Investigators screened for mutations without knowledge about the background (e.g., sex, age, clinical features, and family history) of the patients. The study protocol complied with the Declaration of Helsinki and was approved by the Committee on the Ethics of Human Research of the Tokyo Metropolitan Institute of Gerontology and by similar committees of participating hospitals; and written informed consent was obtained from each participant.

2.3. The suspension array-based mtDNA mutation detection system

We established an array-based screening system for the detection of the 61 representative mtDNA point mutations. To screen for mtDNA mutations, we employed the suspension array system using the analyzer Luminex 100 (Luminex Corp., Austin, TX) based on the principle of flowmetry. The mutation detection process is depicted in Fig. 2.

Fig. 2.

Fig. 2.

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Flow diagram of the process and the time course of the extensive suspension array-based mtDNA point mutation detection system.

2.3.1. Step 1. Multiplex-polymerase chain reaction (PCR) to amplify the 5′ -biotinylated products

We performed multiplex-PCR to amplify 5′-biotinylated products containing multiple segments of mtDNA containing mutation sites (Fig. 2, step 1). The reaction mixture (25 μL) contained 1 ng of total DNA, 5 pmol of each primer set of multiplex-PCRs (Supplementary Table 3), 0.2 mmol/L of each deoxynucleoside triphosphate, 2 mmol/L MgCl2, and 1 unit of Taq DNA polymerase (Roche Diagnostics, Alameda, CA) in DNA polymerase buffer. PCR amplifications were performed with an initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 94 °C for 20 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 7 min.

2.3.2. Step 2. Hybridization of the 5′-biotinylated multiplex-PCR products to the SSO probes

The system uses 5.6-μm polystyrene color-coded beads, microspheres (Luminex), which are labeled with two spectrally distinct fluorochromes (Dunbar, 2006). By mixing and matching the 10 grades of intensities of each dye, a total of 100 (10 × 10) microspheres, each with a unique spectral signature, which is identifiable by the unique combinations of red and infrared fluorescent emissions after laser excitation. Thus, when microspheres are passed through a capillary in the analyzer, the 1st laser excites the internal fluorescent dyes that allow identification of each microsphere particle against the 10 × 10 two-dimensional fluorescent matrix (microsphere addresses). These microspheres were combined into a set. Up to 100 different microspheres can be identified simultaneously in a single reaction vessel of a 96-well plate. Microspheres were covalently coupled with SSO probes (Supplementary Tables 4 and 5) to detect mutant or wild-type mtDNA. For this purpose, the synthesized mutant-specific or wild-type-specific SSO probes were coupled onto carboxylated microspheres through a carbodiimide-base coupling procedure (Dunbar, 2006). To discriminate single nucleotide changes in the PCR products, we performed direct hybridization between the biotinylated multiplex-PCR products and the SSO probes on the surface of the microspheres (Fig. 2, step 2). The mixture of the microspheres coupled with SSO probes and biotinylated amplicons was heat-denatured at 95 °C for 2 min and then hybridized at 52 °C for 30 min in buffer.

2.3.3. Steps 3–5. Streptavidin-R-phycoerythrin binding and calculation of median fluorescence intensity (MFI) values

After hybridization, microspheres were washed three times at room temperature, and incubated for 15 min in a solution with a reporter fluorochrome, streptavidin-R-phycoerythrin (SA-PE, Moss Inc., Pasadena, MD), which binds to biotinylated DNA strands at 52 °C (Fig. 2, step 3). After washing, microspheres were subjected to flowmetry with a Luminex 100 (Fig. 2, step 4). The 2nd laser excited SA-PE, and the fluorescence intensity was quantified; while the 1st laser excited the microsphere internal dyes to identify each microsphere. For each probe, at least 50 microspheres were counted, which represented 50 replicate measurements (Ye et al., 2001), then MFI value was calculated and reported by using Luminex 1.7 software (Dunbar, 2006; Ye et al., 2001). The MFI value represents the median signal intensity measured per bead set, which provides a reference value for each bead analyzed in a sample run (Diaz et al., 2006). The MFI value is preferred over mean fluorescent value because mean values can be distorted by outliers, whereas median values remain consistent in the presence of outliers.

Because mtDNA mutations can be homoplasmic or heteroplasmic, it was important to evaluate simultaneously MFI values from two corresponding alleles (mutant and wild-type) for each mutation site in the same sample. The two analyses distinguish heteroplasmic versus homoplasmic states through two-dimensional plots of pairs of MFI values in a scatter diagram with wild-type MFI values on the abscissa and mutant MFI values on the ordinate (Fig. 2, step 5).

2.4. Cut-off values of the mtDNA mutation detection system

Whenever patient DNA samples were measured, at least 10 normal control DNA samples were measured to determine the cut-off MFI values of each mutation detection system. These cut-off values were set by the addition of 3 standard deviations (SD) to the mean MFI values as background levels (mean + 3 SD). Positive MFI values were those above the cut-off values; and negative MFI values were those below the cut-off values. When we detected both mutant and wild-type signals, we considered the mutations to be heteroplasmic. In contrast, when we detected only mutant signal intensities, we considered the mutations to be homoplasmic. However, we could not define cut-off MFI values of each wild-type detection system, because the numbers of patient DNA samples were limited. To confirm the mtDNA point mutations detected by this method, we sequenced the putative mutation sites by standard procedures (Nishigaki et al., 2003; Ueno et al., 2009) and compared the sequences with the revised Cambridge reference sequences (Anderson et al., 1981; Andrews et al., 1999). To compare MFI values obtained by the m.3243G detection system with the mutation loads, we performed last-cycle PCR-restriction fragment length polymorphism (RFLP) analysis on the mtDNA.

2.5. 5′-Biotinylated primers and probes for the suspension array-based system

To amplify mtDNA fragments using appropriate sets of 5′-biotinylated primers, we carried out two sets of multiplex-PCR: one was an 11-plex PCR for Series A, and the other was a 10-plex PCR for Series B. First, primers used to generate the amplicons were carefully designed to exclude mitochondrial polymorphic sites located at least seven bases from the 3′ ends of the primer sequences when possible (Supplementary Table 3). Validity of 11-plex PCR for Series A and 10-plex PCR for Series B were verified through examination of over 3000 and 400 DNA samples, respectively.

Second, we provided multiple SSO probes to detect both mutant and wild-type genotypes of mtDNA. Thus, we carefully selected and designed a total of 118 SSO probes (65 for Series A and 53 for Series B), considering the various mtSNPs of Japanese in our mtSNP database, as shown in Supplementary Tables 4 and 5. When mtSNPs were present in the vicinity of target mutations, we used two pairs of SSO probes that matched the mutant and wild-type with or without the polymorphic sites. For instance, (1) the detection of the m.12706T>C mutation in the MT-ND5 gene causing MILS mutations (Taylor et al., 2002) can be inhibited by the presence of mtSNP m.12705C>T, which is found in haplogroups A and N9a as well as in macrohaplogroup M among Japanese (frequency, 81%). Nevertheless, the m.12706T>C mutation is expected to be detected even in the presence of mtSNP m.12705C>T, because four probes were designed to detect the mutant and wild-type with or without the polymorphism. (2) Four probes were designed for the analysis of the m.1606G>A mutation in the MT-RNR1 (12S ribosomal RNA) gene causing ataxia, myoclonus, and deafness (AMDF; Tiranti et al., 1998) to avoid interference by the mtSNP m.1598G>A, which is found in haplogroups B5b, N9b1, and N1b in Japanese (frequency, 4.2%). (3) Similarly, for the detection of the m.15498G>A mutation in the MT-CYB (cytochrome b) gene (Andreu et al., 1999), two pairs of probes were designed to avoid interference by the mtSNP m.15497G>A, which is characteristic of haplogroup G1a in Japanese (frequency, 3.7%). Therefore, the present system can be used for the screening of various mtDNA point mutations, even in the presence of polymorphisms, at least in an East Asia population. First, we evaluated a total of 69 SSO probes for detecting mutations (37 for Series A and 32 for Series B) for specific hybridization to 5′-biotinylated synthesized oligonucleotides. Second, we verified the detection system using suspension array technology with 79 DNA samples from patients harboring previously reported mtDNA mutations.

3. Results

3.1. Extensive rapid detection of 61 point mutations in mtDNA of patients

First, the two sets of multiplex-PCRs, an 11-plex PCR for Series A and a 10-plex one for Series B, were completed, which verified the detection systems for 20 of the 61 mutations with 79 DNA samples from patients with mitochondrial diseases due to mtDNA point mutations, as shown in Fig. 3. Next, target point mutations in the mtDNA and mtSNPs in the vicinity of the mutations were confirmed by DNA sequencing.

Fig. 3.

Fig. 3.

Fig. 3.

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Scatter diagrams with the mutant MFI values on the y-axis and wild-type ones on the x-axis. Open rhombuses indicate MFI values of mutation-positive DNAs. Asterisks in ‘‘j” and ‘‘n” indicate MFI values of cybrid DNAs with m.8344A>G in the MT-TK gene and m.8993T>G in MT-ATP6 gene, respectively. Broken lines indicate cut-off MFI values. One DNA sample was not positive with either the m.8993T (wild-type) or the m.8993G (mutant) probe, because this sample harbored the m.8993T>C mutation, as confirmed by sequencing analysis (n). In contrast, both signal values of m.8993T (wild-type) and m.8993C (mutant) of six samples were negative, because they harbored the m.8993T>G mutation, as confirmed by sequencing (m). Similarly, the signals of both m.9176T (wild-type) and m.9176C (mutant) in five samples, four of which were from the same Japanese maternal family, were negative; because they harbored mtSNP m.9181A>G in MT-ATP6 gene, which was confirmed by sequencing (o). As mtSNP m.11782C>T was confirmed by sequencing, both signals of m.11778G (wild-type) and m.11778A (mutant) of two samples from Taiwanese individuals were negative (p). As the mtSNP m.11782C>T is rare among Japanese, it had not been registered in the human mtSNP database. The signals of both m.13513G (wild-type) and m.13513A (mutant) in two samples were negative, because they harbored mtSNP m.13506C>T in their MT-ND5 gene, as confirmed by sequencing (r). Because the mtSNP m.13506C>T is rare in Japan, it also had not been registered in the human mtSNP database. Although the background MFI values of mutation detection systems for m.3255A>G (c) and m.12315A>G (q) were higher than those of the other mutation detection systems, we could clearly detect each mutant DNA by these detection systems.

The following numbers of samples were considered almost homoplasmic: one each for the m.1555A>G, and m.8993T>C mutations; six for the m.9176T>C mutation; five for the m.11778G>A mutation; and one for the m.14709T>C mutation. In these samples, we detected positive signals only for the mutants (Fig. 3).

By using the mtDNA point mutation detection system of Series A (29 mutations) and Series B (32 mutations), we screened a total of 3103 mutant sites in 107 DNA samples for Series A and 13,101 mutant sites in 397 DNA samples for Series B, which were provided from participating hospitals and institutions not only in Japan but also in the USA, Taiwan, and France. Importantly, we succeeded in determining 99.4% (Series A) and 99.6% (Series B) of the targeted mutant sites by use of the system. Only 64 (0.4%) of mutant sites in 45 DNA samples could not be determined, because both wild-type and mutant signals were negative. In those instances, we determined the sequences of the DNA fragment flanking the putative mutation sites, and found rare polymorphisms within the binding sites for both the mutant-specific and the wild-type-specific probes.

3.2. Detection of m.3243A>G mutation by the suspension array system

We analyzed 22 DNA samples (blood, saliva, skeletal muscle, and urine sediments) with the m.3243A>G mutation in the MT-TL1 gene from 14 MELAS or MIDD patients (Fig. 4). All DNA samples with the m.3243A>G mutation yielded positive MFI values for both m.3243A (wild-type) and m.3243G (mutant), which were consistent with heteroplasmy. None of the control samples gave a positive signal for m.3243G. The MFI values of m.3243G were significantly different (P < 0.01) between the 22 samples with m.3243A>G (MFI 9740 ± 1370, mean ± SD) and the 44 control samples (MFI 1200 ± 160, mean ± SD), as assessed by the Student’s t test. We also assessed the exact percentage of mutation of these blood DNA samples by last-cycle PCR–RFLP analysis.

Fig. 4.

Fig. 4.

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Scatter diagram of MFI distributions of both m.3243A and m.3243G detected by the suspension array technique. Twenty-two samples (blood, saliva, and urine sediments) from patients with the m.3243A>G mutation (MFI 9740 ± 1370, mean ± SD) and 44 control samples from patients with the other mitochondrial diseases (MFI 1200 ± 160, mean ± SD) were tested. Double asterisks indicate a significant difference (P < 0.01) between samples from MELAS patients and controls assessed by Student’s t test. The diagram shows MFI values for the mutant on the y-axis and those for the wild-type on the x-axis. Broken lines indicate the cut-off MFI value of m.3243G (mutant).

To verify the detection level of the m.3243A>G detection system, we evaluated the relationship between mutation loads and signal intensity values (MFI 5570 ± 952, mean ± SD) in 16 blood DNA samples with a relatively low mutation load of the m.3243A>G (8.4% ± 5.6, mean ± SD; range of 2–23%). Pearson’s correlation coefficient (R) and the corresponding P value for this correlation were R = 0.82 and P < 0.001, respectively. We estimated that the minimum detection limit of the system was approximately a 2% mutation load (Fig. 5).

Fig. 5.

Fig. 5.

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Correlation between MFI distribution of m.3243G estimated by the suspension array system and m.3243G mutational load assessed by RFLP analysis with GeneMapper software software Version 4.0 (Applied Biosystems, Foster City, CA). The low mutational load (8.4% ± 5.6, mean ± SD; range of 2–23%) of m.3243A>G blood DNA in 16 samples (MFI 5570 ± 952, mean ± SD) was tested. Pearson’s correlation coefficient (R) and the corresponding P value for this correlation were R = 0.82 and P < 0.001, respectively. The curved line approximation is given in the diagram. The broken line indicates the cut-off MFI value for m.3243G. The boxed number indicates the minimal detection limit.

4. Discussion

According to the MITOMAP database, more than 200 point mutations in mtDNA have been associated with mitochondrial diseases. Therefore, the diagnosis of these diseases is complex. The most common and well-established high-sensitivity method for detecting mtDNA mutations is the last-cycle PCR–RFLP using 32P. With this method, the mtDNA mutations must be tested individually; although clinicians usually request screening for several mutations. For the detection of less common or novel mutations, conventional DNA sequencing is necessary. However, the entire mtDNA sequence must be determined for each patient. All the mutations in mtDNA must be confirmed by the human eye. In addition, mutations must be distinguished from a multiplicity of polymorphisms. Since most pathogenic mtDNA mutations are heteroplasmic, it is difficult to detect mutations present at low levels of heteroplasmy by conventional sequencing.

Here, we reported an extensive and rapid screening system using suspension array technology for the detection of mitochondrial diseases due to mtDNA point mutations. Our detection system has three potential advantages over standard RFLP and direct DNA sequencing techniques. First, it makes it possible to screen for 61 different mtDNA point mutations (29 for Series A and 32 for Series B), as was shown in Fig. 1. Second, this method is available to detect heteroplasmic mutations. The 22 samples with m.3243A>G revealed positive signals with both mutant- and wild-type-specific probes in this detection system, in which the detection limit was approximately 2%. The clinical phenotypes of patients with m.3243A>G include not only MELAS but also mitochondrial diabetes, and this mutation is thought to be involved in as many as 1% of all patients with diabetes mellitus (DM) in Japan (Kadowaki et al., 1994). This type of DM progresses more rapidly than other types of DM (Suzuki et al., 2003), and is more resistant to usual therapies, thus making early diagnosis particularly important. Third, this method is rapid and suitable for large-scale screening, because we can use universal 96-well plates for DNA samples from 96 individuals; and analysis of each plate can be completed within 1 h by using the suspension array system (Fig. 2). For conventional sequencing of the entire mtDNA, our average cost, excluding the hardware and labor costs, is approximately 100 US dollars per DNA sample. Usually, it takes several days to obtain the results by the conventional sequencing. By our mutation detection system, we estimate our average cost, excluding the cost for purchasing the suspension array system and operator costs, to be less than 0.25 US dollar per mutation, including the cost of generating the multiplex-PCR amplicons. Thus, it cost less than 20 US dollars for analyzing the 61 mtDNA mutations for each DNA sample, and all of the results were obtained within 1 day. We think this system will be helpful not only to medical staff but also to patients, as it saves both the time and the costs of genetic screening.

According to recent epidemiological studies, subjects with m.3243A>G including asymptomatic cases in the general population are thought to be as common as 6.57–16.3 per 100,000 (Chinnery et al., 2000; Majamaa et al., 1998; McFarland et al., 2002; Schaefer et al., 2004). Manwaring et al. (2007) reported that the population prevalence was much higher, at 236/100,000. Similarly, Vandebona et al. (2009) reported that the m.1555A>G mutation affects about 1 in 500 subjects in a European population-based cohort screened by RFLP techniques. The prevalence of patients with mitochondrial diseases in the general population is still not precisely defined. For such large-scale screenings for multiple representative mtDNA point mutations, our detection method would be both suitable and rapid.

At present, our detection system is customized for the Japanese, because we designed the multiple patterns of SSO probes based on mtSNPs common to the Japanese people. In this study, in two samples from Taiwan, signals of both the m.11778G (wild-type) and the m.11778A (mutant) were lower than the cut-off signal values; because they contained the mtSNP m.11782C>T, which is only four nucleotides from m.11778. According to our human mtSNP database, this mtSNP is one of the characteristic polymorphisms of mitochondrial haplogroup Z, which is relatively frequent among Taiwanese, but which is relatively rare in the Japanese population. Therefore, it will be necessary to consider the presence of haplogroup Z-specific polymorphism, m.11782C>T, when we analyze the prevalence of the m.11778G>A mutation in South Asian countries.

In conclusion, we have constructed an extensive and rapid screening system for the detection of mtDNA point mutations by using Luminex suspension array technology. With it we detected 61 kinds of mtDNA pathological point mutations. We believe this genetic screening platform, based on high-throughput mtDNA mutation detection, is useful to reach a definitive genetic diagnosis of mitochondrial diseases.

Supplementary Material

Suspension Array mtDNA Supplementary Material

Acknowledgments

The authors wish to express great thanks to Saba Tadesse, Yukiko Abe, Kazumi Murakami, and Hideki Okada for their helpful discussions and excellent technical support. This work was supported in part by a grants from the program Grants-in-Aid for Scientific Research (C) [18590317 to Y.N., and 21590411 to H.H.]; by Grant 20B-13 from the program Research Grants for Nervous and Mental Disorders of the Ministry of Health, Labour, and Welfare (to M.T.); and by grants for scientific research from the Takeda Science Foundation (to Y.N. and M.T.), Sankyo Foundation of Life Science (to Y.N.), the Kao Research Council for the study of Healthcare Science (to Y.N.), and the Marriott Mitochondrial Disorder Clinical Research Fund (to S.D. and M.H.).

Footnotes

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mito.2010.01.003.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Suspension Array mtDNA Supplementary Material
NIHMS1630799-supplement-Suspension_Array_mtDNA_Supplementary_Material.doc (311KB, doc)

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