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Articles |
1
Molecular Medicine Unit-INGO (Sergas), University of Santiago de Compostela, Hospital de Conxo, 15706 Santiago de Compostela, Spain.
a Address correspondence to this author at: Unidad de Medicina Molecular-INGO, Hospital de Conxo, Rua Ramón Baltar s/n, 15706 Santiago de Compostela, Spain. Fax 34-981-951679; e-mail apimlbar{at}usc.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We studied 16 patients with HNPP, 4 with CMT1A, and 49 control subjects. We used real-time PCR on the LightCycler system with use of a single capillary tube and no post-PCR handling. A polymorphic fragment of the PMP22 gene was amplified to determine gene dosage for heterozygous samples. The presence of two alleles was used to indicate that no deletion was present in HNPP samples. The ratio obtained between the areas under each allele melting curve of heterozygous CMT1A samples was used to determine whether the sequence was duplicated or normal. Homozygous samples required a competitive gene dosage test, where the ratio between the areas under the melting curves of the target DNA of samples and of the competitor molecule was used to determine whether the target sequence was duplicated, deleted, or normal. Samples from HNPP, CMT1A, and controls were analyzed.
Results: Area ratios were 
0.6, 1.0, and 2.0 for HNPP, control,
and CMT1A samples, respectively. The results agreed with those obtained
by Southern blotting and microsatellite analysis in the same samples.
Conclusions: Direct and competitive real-time fluorescent PCR can differentiate one, two, or three copies of the target DNA. The method described is sensitive and accurate for detection of CMT1A duplications and HNPP deletions and is faster and easier than current methods.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CMT1A is associated with a tandem DNA duplication of a specific 1.5-Mb region at chromosome 17p11.2-p12 (1)(2), whereas HNPP is associated with a deletion of the same region (3). The peripheral myelin protein 22 (PMP22) gene is located within the 1.5-Mb CMT1A monomer (4)(5)(6)(7)(8). Although mutations within the PMP22 gene have been identified in some cases of CMT1A and HNPP, a gene dosage effect has been proposed as the principal underlying pathomechanism for development of CMT1A and HNPP (9)(10)(11).
Molecular diagnosis of CMT1A and HNPP involves detection of the DNA duplication or deletion located at the 17p11.2-p12 region. Southern blots and pulsed-field gel electrophoresis are the methods currently used to assess gene dosage (12). Other methods include fluorescence in situ hybridization (13) and analysis of polymorphic markers located within the duplicated/deleted region (14)(15)(16). However, these methodologies for the diagnosis of CMT1A and HNPP have several disadvantages. The hybridization-based techniques are time-consuming, and large amounts of high-quality DNA are needed. The more specialized fluorescence in situ hybridization assay is available from a limited number of clinical laboratories. Indirect methods based on microsatellite or single nucleotide polymorphic markers analysis depend on the informativeness of the marker and could lead to a wrong interpretation of data.
Recently, PCR-based quantitative strategies have also been developed (17)(18). In these assays, an internal standard of disomic copy that is located outside the region of interest is coamplified with the target DNA. The ratio of the amount of both PCR products indicates whether there is duplication, deletion, or no change in the target DNA. Although very good results are obtained with these strategies, the choice of internal standard is still a critical aspect to overcome. It is widely known that competitive PCR is the most suitable method of quantification when highly accurate determinations are required, e.g., mRNA quantification in gene expression (19)(20)(21)(22). In this method, a known copy number of a competitor is introduced directly into the PCR mixture with the target DNA. The competitor, which is almost identical to the target DNA, is amplified with the same sets of primers. Therefore, the efficiency of amplification for the two amplicons is the same. However, it is necessary to construct calibration curves of different competitor concentrations. Moreover, sample dilutions must be performed to ensure that sample measurements fall within the range of values set by the calibration curve. Finally, the construction of the competitor, which must be distinguishable from the target sequence by a difference in size or sequence, is a critical factor for reliable quantification of the amplified products.
In this study, we describe a rapid strategy to determine gene dosage in CMT1A and HNPP patients based on real-time fluorescent PCR. In the first step, a fragment of the PMP22 gene is amplified. The PMP22 fragment presents an intronic polymorphism at position +31(C/T), described at position +33 by Haupt et al. (16). The PCR products are genotyped by analyzing their melting temperatures, and one or two different melting curves are obtained, corresponding to homozygous or heterozygous genotypes, respectively. Heterozygous samples are diagnosed directly: in HNPP individuals a deletion is discarded; in CMT1A samples, the ratio between the areas under the melting curves determines relative concentrations for both alleles and reveals whether the individual has a duplication. Homozygous samples need a subsequent step of competitive real-time fluorescent PCR. The fragment of the PMP22 gene and a competitor are coamplified with the same set of primers. The competitor is identical to the fragment of PMP22 but differs in only one nucleotide at the site of the polymorphism. After the coamplification a melting analysis is performed, and two different melting curves are obtained, one for the competitor and the other for the target DNA of the sample. Again, the ratio between the areas under the melting curves determines relative concentrations for both target DNA of the sample and competitor, and reveals whether the target sequence is duplicated, deleted, or normal.
For this competitive gene dosage analysis, two competitors were constructed and two genomic controls were used. Competitors and controls were diluted to a concentration that gave exactly the same area under the melting curve after the PCR and melting analysis.
Because all samples were analyzed using the same competitor dilution, they were adjusted to the concentration of the genomic control. For this purpose, a calibration curve was prepared by amplification in the LightCyclerTM (Roche Molecular Biochemicals) of a single-copy gene, the tissue plasminogen activator (TPA), which was used as reference. Relative quantification of samples in the LightCycler provides the sensitivity and accuracy required (23)(24)(25).
The proposed method is the most rapid reported to date. Genotyping and direct gene dosage assay of the samples take 20 min. Once the concentrations of the samples are adjusted to the genomic control concentration (20 min), the whole competitive gene dosage takes an additional 20 min. The analysis is performed in a single capillary tube, avoiding cross-contamination and post-PCR manipulation. Furthermore, the detection in a single system without further electrophoresis makes the system suitable for large-scale high-throughput sample analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Controls were 49 healthy individuals from a random population, carrying two copies of the CMT1A region.
genotyping and direct gene dosage assay
A 239-bp fragment including exon 3 and the intron-exon boundaries
of the PMP22 gene was amplified using the primers described
by Nicholson et al. (27). The probes hybridize to the
PMP22 polymorphic site internal to the PMP22
primer pair. The sequence of the sensor fluorescein-labeled probe was
5'-TTCCAAATTCTTGCTGGTAAGTTGTGGAT-3', and the sequence of the anchor
LC-Red 640-labeled probe was 5'-TAAAGTCCATGTGGAAGCGGGGT-3'. The
adjacent probes emit fluorescence only while they are hybridized to
their complementary strand. Amplification was performed using the
LightCycler DNA Master Hybridization Probes reagent set (Roche
Molecular Biochemicals) in a standard PCR containing 0.5 µmol/L of
each primer and 0.1 µmol/L each probe in a 20-µL final volume with
2 µL of sample. A negative control without DNA sample was included in
all assays. The reaction mixture was denatured at 95 °C for 2 min,
followed by 31 cycles of denaturation (95 °C for 0 s; ramp
rate, 20 °C/s), annealing (55 °C for 10 s; ramp rate,
20 °C/s), and extension (72 °C for 5 s; ramp rate,
20 °C/s). After amplification of the PMP22 gene fragment,
the melting curve was determined by holding the reaction at 55 °C
for 10 s and then heating slowly to 94 °C with a linear rate of
0.2 °C/s while the fluorescence emitted was measured. Melting curves
were generated by plotting fluorescence (F) vs temperature
(T). Discriminated melting peaks were produced by the
LightCycler software. The melting peaks were plotted as the rate of
change of fluorescence (-dF/dT) vs T. The areas
under the melting curves were calculated using the LightCycler software
package. In all experiments, an unaffected control was included, the
ratio of the areas under the melting curves for the control was
normalized to a value of 1.0, and the samples were scaled accordingly.
All assays were carried out in duplicate, and the average value was
used.
competitive gene dosage assay
Construction of competitors and calibration of their melting
curves.
The 239-bp PMP22 fragment containing exon 3 and
the exon-intron boundaries was amplified from DNA samples of two
homozygous individuals (C/C and T/T) for the +31(C/T) polymorphism. PCR
was carried out in a 50-µL reaction containing 2.5 mmol/L
MgCl2, 0.8 mmol/L dNTPs, 0.5 µmol/L each primer
described above, 2.5 U of Taq High Fidelity (Roche Molecular
Biochemicals), and 2 µL of template DNA. The reaction was 30 cycles:
94 °C for 45 s, 59 °C for 60 s, and 72 °C for
90 s, with a final extension of 72 °C for 7 min. PCR products
were purified using MicroSpin S-300HR columns (Amersham Pharmacia
Biotech) and were 10-fold serially diluted in Tris-EDTA (10 mmol/L
Tris, pH 8.0, 1 mmol/L EDTA) to 10-8. The
diluted products were stored at -40 °C until use.
Two types of competitors were obtained, one carrying the polymorphic allele +31T, designated the polymorphic standard (Ps), and the other the normal allele +31C, designated the wild-type standard (WTs). Two different unaffected genomic controls were also used: one homozygous +31 (T/T) for the intronic polymorphic site of PMP22 gene, which was coamplified with the competitive WTs, and other homozygous +31(C/C) for the polymorphic site, which was coamplified with the Ps.
After coamplification of the genomic controls with serial dilution from 10-5 to 10-8 of their respective competitor, the melting curves were generated. The dilution of competitor that gave equal areas under the melting curves for both products, genomic DNA and competitor, was chosen to be further coamplified with the target DNA in the samples. The same dilutions of competitors and genomic controls were used in all subsequent experiments.
Adjustment of DNA concentrations of samples to the genomic
control.
To adjust the DNA concentration of samples to the
concentration of the appropriate genomic control, a concentration
calibration curve was prepared. For this purpose, a fragment of a
single-copy gene outside the region of interest, the TPA
gene, was amplified in five serial dilutions of each sample DNA (1:1,
1:5, 1:10, 1:50, and 1:100). The previously diluted genomic controls
were also amplified. The amplification was performed in the LightCycler
system using the LightCycler DNA Master Hybridization Probes reagent
set. Sequences of the primers were as follows: forward,
5'-CGACAATGACATTGGTAAGAGCTCG-3'; and reverse,
5'-AGGCCCGTGTGTGTAAACATAGGTG-3'; primers were added to the PCR
reaction mixture to a final concentration of 0.5 µmol/L. The sensor
and anchor fluorogenic probes were, respectively,
5'-CCTCCTCCTCCAGCCCCTGCCC-3' for the fluorescein-labeled probe, and
5'-TCCTGCGTGTTCCTCCCCTCCC-3' for the LC 705-labeled probe. Each
probe was added to a final concentration of 1 µmol/L. The cycling
program consisted of a initial denaturation at 95 °C for 2 min and
45 cycles of 95 °C for 0 s, 65 °C for 10 s, and
72 °C for 5 s, with a ramp rate of 20 °C/s. Negative
controls (without template DNA) were included in the assay, and the
test was performed in duplicate.
The reproducibility of the calibration curve was analyzed by evaluating the slope and the correlation coefficient of the curve in each experiment. By extrapolation within the calibration curve, the dilution of the sample with the same concentration of its corresponding genomic control was chosen.
Competitive gene dosage real-time fluorescent PCR.
Competitive
PCR conditions were basically the same as described above for the
real-time fluorescent PMP22 amplification with the following
difference: 1 µL of competitor was added together with 1 µL of
target DNA to the 20-µL final volume of the PCR mixture.
After amplification, the PCR products were subjected to a temperature transition from 50 °C to 94 °C, and the melting curves for the competitor and the target DNA of the samples were generated in the LightCycler. The ratio of the areas under the melting curves from the competitor and the target DNA was calculated for each sample. All assays were carried out in duplicate. As in the direct gene dosage test, all experiments included an unaffected control. The ratio of the areas under the melting curves for this control was normalized to a value of 1.0, and the samples were scaled accordingly.
determination of variability of pcr assay
All samples were tested in duplicate, and the average of the two
ratios from each sample was included in an interassay variation
analysis. Intraassay variation was assessed by analyzing five
replicates of a deleted, a duplicated homozygous, a duplicated
heterozygous, a normal homozygous, and a normal heterozygous samples. A
day-to-day variation was determined by conducting analyses of the same
samples, in duplicate, on 10 separate occasions. All data were reported
as the mean, SD, and CV of the results obtained in the different
assays.
Five replicates of a heterozygous normal sample were analyzed in five different assays to determine variability within a PCR and among different PCR assays. This analysis was performed by one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). The genotyping strategy allowed the rapid and direct
identification of heterozygous samples indicating the absence of
deletion.
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Despite the 1:1 relationship of alleles in heterozygous samples, a
ratio between the areas under the melting curves that substantially
differed from 1.0 could be observed. This is attributable to small
changes in PCR conditions and mixtures. ANOVA showed significant
differences among five PCR assays of the same heterozygous sample
(P <0.001; Fig. 2
), but the same sample showed a greater variation among
different PCR assays than among replicates within the same PCR. Because
all samples analyzed in the same PCR showed small deviations, reliable
and accurate results could be achieved by including a normal
heterozygous sample as the control in all assays, and then normalizing
its ratio value to 1.0 and scaling the samples accordingly. The
deviation from the ratio of 1.0 value was negligible (one-way ANOVA,
P = 0.025; Fig. 2
).
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The 16 diagnosed HNPP samples included 3 heterozygous, 10 wild-type homozygous (+31C), and 3 polymorphic homozygous (+31T). The absence of a deletion in the HNPP heterozygous samples was further confirmed by microsatellite analysis and sequencing of PMP22 gene.
The unaffected control group consisted of 49 samples, which included 22 heterozygous, 24 wild-type, and 3 polymorphic homozygous genotypes.
Four CMT1A patients were analyzed: three carried both alleles, and the
other carried only the wild-type allele. The scaled ratio
between the two areas under the melting curve in the heterozygous
samples of CMT1A allowed the correct genotype, +31(C/C/T) duplication
of the C allele, without further analysis (Fig. 1B
).
All genotypes can be quickly determined with unambiguous results. Duplications in CMT1A heterozygous samples [+31(C/T/T) or +31(C/C/T)] and absence of deletions in HNPP heterozygous samples (C/T) can be directly determined. In these samples, the competitive analysis is not needed.
competitive gene dosage assay
After the competitive real-time fluorescent PCR and melting
analysis of samples, three different patterns of melting curve plots
were obtained: one in which the area under the melting curve of the
sample was approximately one-half the area of the competitor (Fig. 3
A), indicating a deletion in HNPP samples; one in which the area
under the melting curve of the sample was approximately equal to the
area of the competitor (Fig. 3B
), indicating normal gene dosage; and
one in which the area under the melting curve of the sample was
approximately double that of the area of the competitor (Fig. 3C
),
indicating the duplication in CMT1A samples. The results were similar
when either competitor Ps or WTs was used.
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Adjustment of the DNA concentration of all samples to the concentration
of the genomic control that gave an area under the melting curve equal
to the area of the competitor was required before the competitive
real-time fluorescent PCR was performed. An example of amplification of
the TPA gene in serial dilution of a sample and the
corresponding calibration curve is shown in Fig. 4
. The dilution of each DNA sample that had the same
concentration as the genomic control was used for the competitive
assay.
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assay variation
In the direct analysis, the 22 normal heterozygous samples
analyzed showed a mean ratio of 1.066 (SD = 0.137) and an
among-sample variation 13% (Fig. 5
). The three duplicated heterozygous CMT1A samples, with a mean
ratio of 2.108, showed a 4.5% interassay variation (SD = 0.095;
Fig. 5
).
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The intraassay variation was 2.8% (mean ratio = 0.996; SD = 0.028) for the normal heterozygous sample, and 7.7% (mean = 2.146; SD = 0.164) for the CMT1A heterozygous sample. This result and the 99% confidence interval (1.891–2.326) were clearly separated from the 99% confidence interval for normal heterozygous samples (0.983–1.148). Thus, any potential misdiagnosis was avoided.
In the competitive analysis, the intraassay variation was 15%
(mean ratio = 0.633; SD = 0.094) for the HNPP samples, 13%
(mean = 1.002; SD = 0.132) for the normal samples, and 6.6%
(mean = 1.904; SD = 0.126) for the homozygous CMT1A sample
(Fig. 6
). The interassay variation was 17% (mean = 0.559; SD
= 0.093) for 13 HNPP samples, 19% (mean = 0.954; SD = 0.184)
for 24 normal samples, and 9.3% (mean = 1.82; SD = 0.169)
for the homozygous CMT1A sample (Fig. 5
). The results were similar to
those of the direct analysis, and the three ratio value conditions
(normal, deleted, and duplicated) were clearly separated at the 99%
confidence interval.
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Small differences in ratios could be observed among different PCR
assays. We therefore extended the intraassay analysis, conducting the
test over 10 separate occasions. The samples, analyzed in duplicate,
were normalized to a normal control, and the mean of the 10 averaged
ratios was obtained (Table 1
). The day-to-day variation of each sample was negligible and
similar to that of the interassay.
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As a result of the reported data, we used the following criteria in the analysis/interpretation of the competitive gene dosage test: Samples were diagnosed as normal if the areas under their melting curve were within a 10% interval of the competitor area (sample/competitor ratio of 0.90–1.20 after normalizing to the control and scaling). A deletion was diagnosed when the sample/competitor ratio was within a 15% interval (0.53–0.72), and a duplication was diagnosed when the target/competitor ratio was within a 10% interval (1.71–2.10).
In the direct dosage test, the same confidence intervals (0.90–1.20 for normal samples and 1.71–2.10 for duplicated samples) were used.
The results in cases with ratios outside the confidence intervals were considered inconclusive. The use of these criteria reduced the probability of misdiagnosis. With the application of these criteria, almost all samples were well diagnosed. Only 5 of 66 samples led to inconclusive results, and their analyses were repeated. A careful analysis of the results of these samples revealed that all of the problems were attributable to handling, and the subsequent assays permitted a correct diagnosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The melting analysis approach overcomes the aforementioned difficulty.
Sequence-specific fluorescent probes accurately identify two fragments
differing by one single nucleotide (30). In our strategy, we
amplified by real-time fluorescent PCR a PMP22 fragment that
contains an intronic polymorphism (+31C/T), and the sensor probe used
spans the polymorphism. Our results demonstrate the correlation between
gene dosage and ratio of fluorescence signal after melting analysis.
The heterozygous cases, for which this dose is known, confirm this
result. In a single PCR, the ratios of all samples showed a similar
deviation from 1.0 when the area ratios were calculated normalized to a
control. The use of a known unaffected heterozygous sample as reference
allowed us to obtain a ratio value that was independent of the PCR
conditions. Therefore, the test offers a rapid and direct diagnosis of
heterozygous CMT1A samples. For heterozygous samples with duplication
of one allele [+31(C/C/T) or +31(C/T/T)], different areas under both
melting curves are expected (Fig. 1B
). PMP22 genotyping also
offers a rapid and direct method of screening for the diagnosis of HNPP
because HNPP patients heterozygous for this polymorphism would not
carry the deletion. This case illustrates the principle of the method;
the relationship between the different fragment doses determines the
ratio between the areas after melting analysis when the optimized
protocol described above is used.
In homozygous samples, it is impossible to compare the quantity of both alleles, but we can add the reference allele as a competitor to the sample. Following this principle, we developed a strategy based on a competitive real-time fluorescent PCR in which accurate quantification is provided by the LightCycler instrument. Two types of competitors were produced, one +31T to be coamplified with samples with C at position +31, and the other +31C to be coamplified with samples with T at position +31. In both cases, competitor and target molecules were easily distinguished by their respective melting curves without the need of gel electrophoresis. To obtain both competitors, we amplified homozygous samples (+31C/C and +31T/T), which greatly simplified the competitor construction compared with traditional cloning techniques (31)(32). The competitive real-time fluorescent PCR does not present the problem with data interpretation that occurs in conventional competitive PCR because of heteroduplex formation between single strands of calibrator and target that differ in one single nucleotide (33). In the method described, the fluorescence is measured when a single fluorogenic probes hybridize specifically with its complementary strand.
It is important to determine the amount of starting DNA to assure a 1:1 ratio for genome copy number of the sample and the competitor when starting PCR cycling. Competitive gene dosage analysis can be performed immediately after the DNA concentration is quantified in the LightCycler. Although quantification of DNA samples by fluorometric or other conventional methods is possible, the greatest accuracy of the test is achieved when samples are quantified by real-time fluorescent PCR. We have observed that the most critical point in the competitive PCR is the choice of adequate sample dilution for the analysis. There are several advantages of quantification by real-time fluorescent PCR over quantitative PCR methods that rely on endpoint analysis. Most importantly, the sample input copy number is determined during the exponential phase of the reaction, producing a more accurate estimation of the concentration. In addition, real-time fluorescent PCR eliminates the need for post-PCR processing because the amount of product amplified is monitored in each cycle and visualized without gel electrophoresis. The lack of post-PCR manipulation reduces the risk of cross-contamination and makes the assay suitable for automation.
The reference genomic control is used to titrate the competitor before
use. The competitor sequence is diluted to a concentration that
provides a target sequence copy number equal to that of the reference
genomic control. The concentration of genomic control is arbitrarily
chosen; we have used 10 ng of DNA (3000 genome-equivalents). The
competitor and genomic control concentrations are stable almost 1
month. It is convenient to perform the analysis in duplicate and
essential to adjust the genome copy numbers of samples to those of the
genomic controls before competitive assay. This could be a problem for
high throughput of samples, but the LightCycler is rapid enough to
overcome this limitation. The time required for the analysis and
the number of samples that can be processed simultaneously makes this
diagnostic method faster and more economical than previously reported
methods.
The method we have developed combines the competitive approach with real-time fluorescent PCR technology. The main advantage of the method described here over other strategies (18)(34) is the competitive approach: our dosage test does not use controls located on other chromosomal regions different from the target molecule. As stated previously (33), small differences in amplification efficiency are compounded exponentially. A difference of only 5% in amplification efficiency between two initially equal targets can cause one product to appear to be twice the amount of the other after 26 cycles of PCR (31). The efficiencies vary between reactions and for different experiments, so it may not be possible to obtain similar efficiencies for both control and target markers. This limitation is present even when highly accurate systems such as the LightCycler instrument are used. Experiments carried out in our laboratory with the same described samples to quantify PMP22 using TPA as internal control gave inconclusive results (data not shown). The gene dosage test in the method described is feasible and more accurate than trying to achieve equal efficiency for different fragments.
The real-time fluorescent PCR assay that we have developed allows the rapid differentiation of one, two, or three copies of DNA in samples by comparison of the ratios of the areas under the melting curves of allele/allele (heterozygous samples) or sample/competitor (homozygous samples). Therefore, with the proposed dosage test, large populations can be easily and accurately tested for the presence of deletions and duplications. The principle of the method described could be applied to the diagnosis of any pathology caused by changes in gene dosage, and not just the diagnosis of HNPP and CMT1A diseases.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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