In 1993, Prezant et al1 first identified the association of the A1555G mutation in the 12S rRNA gene of mitochondrial DNA (mtDNA) with mitochondrial deafness (MD). It has been found that this mutation correlated with aminoglycoside-induced and adult onset non-syndromic hearing loss in many families of different ethnic origins,2-5 with a prevalence of 0.5%-2.4% in European sensorineural deafness patients and 7% in Chinese patients.6-8 However, the resulting phenotype varies considerably among matrilineal relatives within families or among different families carrying the A1555G mutation, ranging from severe deafness, to moderate progressive hearing loss or even completely normal hearing. Incomplete penetrance and variable expressivity of hearing loss associated with the mutation A1555G are thought to be due to the interaction between genetic factors, such as nuclear modifier genes or mitochondrial DNA, and environmental factors, such as aminoglycoside antibiotics.9,10 However, mtDNA is crucial for the mitochondrial disease and the copy number of mtDNA has been shown to be associated with cellular function. Cellular phenotypes may be affected by the increased consumption of cellular O2 due to increased mtDNA copy number. With regard to MD, mutant DNA has a replication advantage over wild-type, suggesting a possible correlation between the severity of MD and the mutant copy number.11,12
The Amplification Refractory Mutation System (ARMS) can specifically detect the copy number of fragments containing known mutant loci.13 In this study, in order to detect the copy number of mtDNA A1555G, we combined the ARMS method with real-time quantitative polymerase chain reaction (RT-qPCR) to establish an RT-ARMS-qPCR system for the sequence-specific detection of mtDNA copy number. The copy number of fragments containing the mtDNA A1555G mutation was quantitatively determined and methodologically evaluated. We also investigated the applicability of the RT-ARMS-qPCR system in studies of MD, and the relationship between mtDNA copy number and severity of deafness.
Two groups of deaf patients were recruited in this study, the sporadic deafness group included 66 patients (23 male, 33 female) aged 5-21 years who visited outpatient clinic of the First Affiliated Hospital of Fujian Medical University; and the familial group was selected by a survey of 133 subjects (58 male, 75 female) aged 2 months to 82 years, from eight families. Blood samples were collected from only 89 subjects (maternal subjects: n=60, deaf patients: n=29) due to the absence of some family members. Histories were taken from all the deaf patients, including onset age, predisposing factors, disease severity and previous medication use. Pure tone audiometry was performed in the audiometric room of our hospital. The degree of deafness was determined according to the International Classification of Impairments, Disabilities and Handicaps (ICIDH) issued by the World Health Organization (WHO).14 According to the classification of deafness,5 a patient may be diagnosed with MD if he/she has an mtDNA mutation and presents with no other symptoms.
Extraction of DNA
Venous blood (2 ml) was collected from the subjects and treated with the anticoagulant ethylene diaminetetraacetic acid (EDTA). DNA was extracted from 200 μl of fresh whole blood by a Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions.
Three primers were designed by Express Primer software (ABI Corporation, Foster City, CA, USA) according to the ARMS rationale and the requirements for fluorescence quantitative PCR. The primer set included one common primer (P11), one primer for wild-type DNA (P22) and one primer for mutant DNA (P23). In order to increase the specificity, two mismatched bases (underlined) were added to the 3′ end of P22 and P23. The sequences were as follows: P11: 5′-CGAAGGTGG-ATTTAGCAGT-3′; P22: 5′-CACTTACCATGTTACGA-CTACT-3′; P23: 5′-CACTTACCATGTTACGACTACC-3′.
Preparation of the target DNA fragment
DNA extracted from fresh blood of the patients was confirmed by sequencing to contain the wild-type and the mutant mtDNA A1555G and amplified by PCR. PCR was performed in a volume of 25 μl containing 50 ng sample of DNA, 0.06 mol/L primer, 0.2 mmol/L dNTP, 2.5 μl of 10× PCR buffer, 2 mmol/L Mg2+ and 1 U Taq enzyme (ABI Corporation). Thermal denaturation was carried out on a PE9700 (ABI Corporation) thermal cycler at 95°C for 5 minutes, followed by 35 cycles at 94°C for 60 seconds, 58°C for 60 seconds and 72°C for 60 seconds, with a final extension at 72°C for 10 minutes. The PCR products were detected by 1.5% agarose gel electrophoresis (AGE) (60-80 V, 20-30 mA).
Construction of vectors and calculation of copy number
Quantification PCR from original samples was independently duplicated to exclude potential artifacts. To generate a standard curve for quantification of mtDNA, target fragments were purified from AGE using a gel extraction kit (Promega Corporation). Fragments were ligated into plasmid pGEMT Easy (Promega Corporation), and transformed into Escherichia (E.) coli DH5α competent cells and incubated overnight at 37°C. Recombinants were screened by resistance to ampicillin. Plasmids were extracted and sequenced by the Shanghai Sangon Biologial Engineering Technology & Services Co. Ltd. Absorbance (A) values of plasmids DNA were measured by spectrophotometry (GeneQuant Pro; GE Healthcare, UK) and the mtDNA copy number was calculated using the following formula: (X μg/μl plasmid DNA/3177 (plasmid and target DNA length) ×660) × 6.022 × 1023 = Y molecular number/μl. X represents the concentration of plasmid DNA and Y represents copy number.
Optimization of RT-ARMS-qPCR
Under optimal reaction conditions (data not shown), target DNA plasmids were diluted 1:10 by the double-dilution method and used to prepare standard curves for quantification of unknown templates. PCR reactions were in a volume of 20 μl and contained 1×platinum SYBR Green qPCR super MIX UDG (Invitrogen Corporation, Carlsbad, CA, USA), 500 nmol/L primer (P11, P22/P23) and 4 ng DNA. PCR conditions were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, 61°C for 60 seconds, for 40 cycles. Fluorescent signals were detected by PE7000 (ABI Corporation) and analyzed by its SDS software. The content of mtDNA in the original template was calculated according to the cycle threshold (Ct) values. Melting curve analysis began at 60°C. The reproducibility, specificity, sensitivity and linear range of the RT-ARMS-qPCR technique were evaluated.
The association between the copy number of mtDNA A1555G and the disease severity was analyzed by Spearman's rank correlation test using SPSS 10.0 (SPSS Inc., USA). P <0.05 was considered statistically significant.
Verification of plasmid standards
In order to obtain standard material, fragments containing the mtDNA 1555 locus were amplified and sequenced by PCR using recombinant plasmids as the template. The result of BLAST comparison showed that the sequences of the tested gene fragments were consistent with those reported by Genebank. The sequence of the fragment containing the mtDNA 1555 locus is shown in Figure 1.
Quantification of recombinant plasmid standards
Spectrophotometry showed that the A value for the recombinant plasmid standard containing wild-type mtDNA 1555 was 0.07. Thus, we calculated the concentration of 10 μl plasmid DNA + 56 μl ddH2O to be 1 × 109 copies/μl. Serial dilutions (1 × 103, 1 ×104, 1 × 106, 1 × 107 and 1 × 108 copies/μl) of the wild-type standard plasmid were made. The A value for the plasmid standard containing mutant mtDNA 1555 was 0.06. Serial dilutions (1 × 103, 1 × 104, 1 × 106, 1 × 107 and 1 × 108 copies/μl) of mutant standard plasmid were made.
Methodological evaluation of the RT-ARMS-qPCR system
Analysis of melting curves
No primer-dimers, non-specific PCR products or contamination were detected using the RT-ARMS-qPCR system when the melting temperature of the PCR products was (83.2 ± 0.5)°C. Detailed data are not shown.
Recombinant plasmid containing wild-type mtDNA 1555 was used as a template for six separate, simultaneous measurements. The resultant Ct values were 20.5, 20.8, 21.0, 20.3, 20.6 and 20.8. The mean Ct value was 20.7 and the intra-run CV was 1.21%. Consecutive quantitative PCR determination of the same recombinant plasmid DNA sample over six days generated Ct values of 14.3, 14.6, 15.5, 14.9, 15.6 and 15.0. The mean Ct was 15.0 and the iner-run CV was 1.78%. This suggested that the RT-ARMS-qPCR method had a high reproducibility.
Simultaneous fluorescent qPCR assays were performed on a single sample of plasmid DNA containing wild-type DNA 1555, a single sample of plasmid DNA containing mutant DNA 1555, and double distilled water as templates. P11/P23 and P11/P22 were used as primers. Using P11/P23 as primers, a typical “S”-shaped amplification curve was observed for the plasmid DNA containing mutant mtDNA 1555 and no fluorescence was observed for the others. When using P11/P22 as primers, a typical “S”-shaped amplification curve was observed for the plasmid DNA containing wild-type mtDNA 1555 and no amplification was detected for the others, suggesting an acceptable specificity for this method (Figure 2).
Measurement of linear range
The recombinant plasmid containing the mutant mtDNA 1555 standard was diluted 10-fold by the double-dilution method. Nine concentrations (10-109 copies/μl) of the standard plasmid were chosen for qPCR. When the concentration of the standard plasmid was >108 or <102 copies/μl, the amplification curve did not display a typical “S”-shape and the correlation between standard curves were not favorable. A typical “S”-shaped amplification curve was observed when the template concentration was 102-108 copies/μl.
Quantitative standard curves for DNA fragments containing mutant and wild-type mtDNA 1555 loci
A linear equation of Y=-1.76X + 36.35, and a correlation coefficient of 0.983 were obtained when using serial dilutions of the mutant mtDNA 1555 standard to prepare standard curves. When using serial dilutions of the wild-type plasmid standard to prepare standard curves, the linear equation was Y=-1.66X + 30.76 and the correlation coefficient was 0.985.
Detection of the mtDNA A1555G copy number in sporadic and familial deafness groups
Figure 3 shows the copy numbers for the homoplasmic mutant mtDNA A1555G and related clinical data in the sporadic deafness group. The correlation coefficient between the copy numbers of mtDNA A1555G and the severity of deafness was 0.007 with P=0.989 (two-sided), therefore there was no correlation between the copy number and the severity of hearing loss.
The copy numbers of the heteroplasmic mutant mtDNA A1555G and related clinical data are shown in Table 1. The correlation coefficient between the mutant heteroplasmy and the severity of hearing loss was 0.811. The result was statistically significant (P=0.003; two-sided), so there was a statistical correlation between the mutant heteroplasmy and the severity of deafness. A higher percentage of mutant mtDNA indicated greater severity.
Figure 4 shows the copy numbers of the homoplasmic mutant mtDNA A1555G and related clinical data from eight familial groups. The correlation coefficient between the copy number of mtDNA A1555G and hearing loss was 0.352, with P=0.023 (two-sided), suggesting a correlation. A higher copy number of mtDNA A1555G indicated greater disease severity.
Table 2 shows the relationship between the copy number of heteroplasmic mutant mtDNA A1555G and the severity of deafness in non-syndromic hearing loss (NSHL) patients in the familial groups. The correlation coefficient between the copy number of mtDNA A1555G and degree of deafness was 0.90 with P=0.012 (two-sided), indicating a correlation. A higher percentage of mutant mtDNA, i.e. a greater heteroplasmy of mtDNA A1555G, indicated a greater disease severity.
MD is a mitochondrial disease characterized by maternal inheritance, heteroplasmy, threshold effects and a high mutation rate. The main focus of molecular diagnosis of mitochondrial disorders has been on the detection of mtDNA point mutations. However, identification of a mutation is often not sufficient to explain the clinical phenotype. The proportion of mutant heteroplasmy and the total mtDNA level also play an important role in determining the disease severity. Without considering mutant load in affected tissues, we evaluated patients with MD by the RT-ARMS-qPCR system. Our results showed non-correlation between the severity of deafness and the copy number of mutant mtDNA A1555G in patients with homoplasmic mutations in the sporadic group. This may be due to disparity in genetic background, physical age, and age at which medication use began.9 The severity of deafness was not determined by the copy number of mutant mtDNA. In sporadic deafness patients with heteroplasmic mutations, the percentage of mutant mtDNA was associated with the severity of deafness. A higher percentage of mutant mtDNA indicated more severe hearing loss, therefore, the clinical phenotype of deafness was associated with the percentage of mutant mtDNA.
Analyses of the relationship between the copy number of homoplasmic mutant mtDNA A1555G and clinical phenotype in the seven families carrying the mtDNA A1555G mutation indicated a correlation between the copy number of mtDNA A1555G and the severity of deafness. An increase in the copy number of mutant mtDNA may lead to a greater severity of deafness. The copy number of mtDNA A1555G mutation could be the main determinant of the deafness phenotype.
Data from three families carrying heteroplasmic mutations showed that 1) the use of aminoglycoside antibiotics led to severe deafness in A III1 and B III4; 2) mild hearing loss occurred in B II4 when they reached a certain age; 3) the hearing of A III 3, B III 6 and D II2 was normal. These results suggested that the severity of deafness became greater as the percentage of mutant mtDNA A1555G increased with age. This was consistent with the results for Spanish families reported by Estivill et al.2 It also corresponded to a common characteristic of mitochondriopathies, that symptoms occur and become severe when the energy output cannot meet the minimum requirements for the normal functions of cells, tissues and organs because of the excessive accumulation of mutants.
Analyses of the above results showed that for patients with heteroplasmic mutations in either the sporadic group or the familial group, a higher ratio of mutant mtDNA meant a greater hearing loss. The copy number of mutant mtDNA A1555G was moderately associated with the severity of MD. Nonetheless, different degrees of deafness may be observed in individuals with similar percentage of mutant mtDNA A1555G This may be attributed to the many factors that influence deafness, such as aminoglycoside antibiotics, nuclear genes and mitochondrial DNA.10
In conclusion, we specifically analyzed the association between the severity of deafness and mtDNA 1555 mutation in patients with deafness in sporadic and familial deafness groups using the RT-ARMS-qPCR system. We hypothesized that changes in the quality (Heteroplasmy and homoplasmy) and quantity of mtDNA, especially the ratio of mutant mtDNA to total mtDNA, would be the most important factors in determining the severity of deafness. In addition, the genetic background appeared to be one of the factors affecting the clinical phenotype of deafness. This study may contribute to the ultimate clarification of the diversity of clinical phenotypes of MD. In current phase, no treatment is available to prevent, fix or delay MD when one is identified with A1555G mutation, but if the relationship between the ratio of mutant to wild type mtDNA and the severity of MD is clear, we can predict whether a baby from a deaf pregnant woman would be deaf or normal. Besides this, even if an individual carries mutated mtDNA A1555G, if the ratio of mutant to wild type mtDNA is low enough, the use limitation of aminoglycoside antibiotics is unnecessary.
Bai and Wong15 were the first to detect the copy number of heteroplasmic mutant mtDNA A3243G by the RT-ARMS-qPCR technique, providing a technical platform for the quantitative detection of mtDNA mutations. The key to successful detection by the RT-ARMS-qPCR technique lies in designing appropriate primers and PCR amplification conditions. The ARMS system requires two types of primers: “normal” primers that exclusively bind to the wild-type template, and “mutant” primers that bind only to the mutant template. To enhance the specificity of this test, we changed two bases in the antepenultimate and penultimate positions from the 3′ end, in addition to altering the 3′ terminal base of the primers, to ensure favorable reproducibility and specificity. There was no evidence of non-specific PCR products and contamination. Our results showed that the RT-ARMS-qPCR system quantitatively and accurately measured the percentages of mutant and wild-type mtDNA A1555G. Thus, this system may be applicable for investigations and possibly even gene diagnosis of MD.
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