Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR.
Molecular diagnosis of CMT1A or HNPP involves the detection of the respective DNA duplication or deletion. Although several approaches are available for the detection of duplications and deletions, only a few of these allow the detection of both a DNA duplication and deletion in a single test. Molecular diagnosis of CMT1A and HNPP is commonly carried out by hybridizationbased methods. These include pulsed-field gel electrophoresis (PFGE), followed by Southern analysis to detect either duplication or deletion junction fragments (2, 3,10), or quantitation of dosage changes in restriction fragments (11-14). Other less frequently used methods include microsatellite analysis and two-color fluorescence in situ hybridization (2,15).
The techniques used for the molecular diagnosis of CMT1A and HNPP have several limitations. Hybridization-based techniques are time-consuming, require large amounts of DNA, and use radioisotopes. Fluorescence in situ hybridization is also time-consuming, costly, and requires specific equipment not available in all laboratories. Although microsatellite analysis is a PCR-based technique, successful diagnosis is dependent on the informativeness of the marker(s) used. If the markers used are not informative, the sample will require testing with other markers, if available, or by one of the alternative methods.
We report the development of a rapid PCR-based relative DNA quantitation technique that allows the detection of duplications and deletions in a single reaction and overcomes many of the problems associated with the molecular diagnosis of CMT1A and HNPP. This is a relative quantitation method and, therefore, relies on the inclusion of one or more internal control or reference sequences; quantitation of DNA is relative to this reference sequence of known copy number (16-18). An area from within a potentially duplicated or deleted target region is amplified simultaneously with a disomic reference region in a multiplex PCR system. The ratio of the amount of PCR product generated from each amplification reaction indicates whether there is a duplication, deletion, or no change in the target area. This method is, therefore, based on the observation that the amount of PCR product generated from each site of amplification is proportional to the amount of starting template. Detection of PCR products is carried out on a Gel Scan-2000 (GS-2000) automated fragment analysis system, which provides the sensitivity required for the detection of the single-copy dosage changes found in both CMT1A and HNPP. The sensitivity of this method was further demonstrated by the detection of dosage differences in an X-linked gene, the androgen receptor (AR) gene, between males and females.
A major advantage of this system is that the analysis is carried out within a single tube; therefore, any factors influencing the PCR will affect both reactions equally and will not alter the resulting ratio. A further advantage of this technique is the coupling of electrophoresis with detection in a single system that is suitable for large-scale sample analysis.
Materials and Methods
DNA was extracted from blood samples of unrelated individuals in which the diagnosis of CMT1A (50 individuals) and HNPP (30 individuals) had been determined previously by PFGE (14). PFGE analysis was also performed on 50 unaffected individuals to ascertain the absence of a duplication or deletion at 17p11.2-12.
Oligonucleotide primers (Table 1) were designed to amplify two target sequences [part of the PMP22 gene and EW401 (D17S61)] that lie within the potentially duplicated or deleted target region and one reference sequence (5' untranslated area of NF1 gene at 17811.2). PMP22 and EW401 target amplicons were chosen for analysis because they map to the proximal and distal ends of the 1.5-Mb duplication or deletion region, respectively (Fig. 1). Primers were designed to have similar [T.sub.m] (the temperature at which 50% of double-stranded DNA is denatured) values and sequence composition to allow the use of similar conditions for a multiplex PCR system. In the case of the quantitation of the X chromosome copy number, the region encoding exon 4 (19) of the AR gene was coamplified with the same disomic reference region (NF1) used in the CMT1A and HNPP analysis.
PCR was performed in a 10-[micro]L reaction volume containing 50 mmol/L KCI, 10 mmol/L Tris-HCI, pH 9.0, 1.5 mmol/L Mg[Cl.sub.2], 100 [micro]mol/L of each deoxynucleotide triphosphate, 20 pmol of each primer, 5 ng of DNA template, and 0.5 U Taq polymerase (Perkin-Elmer/ Cetus). PCR was carried out using a model 9600 thermocycler (Perkin-Elmer). An initial denaturation at 95[degrees]C for 5 min was followed by 26 cycles of denaturation (94[degrees]C for 30 s), annealing (58[degrees]C for 30 s), and extension (72[degrees]C for 30 s). PCR cycling was ended with a 10-min (72[degrees]C) extension step. An equal volume of loading buffer (15% Ficoll/blue dextran) was added to each reaction mixture, and 4 [micro]L was electrophoresed on a 5% Long Ranger hydrolink (FML) gel that had been prestained with ethidium bromide (1 g/L). PCR products were detected during electrophoresis by a GS-2000 laser-based automated fragment analyzer (Corbett Research). Detected PCR products were displayed as signal peaks by the RFLPscan program (Scanalytic Software, a division of CSPI). The area under each signal peak was proportional to the amount of PCR product generated. Ratios of the area under each peak (target:reference) were then calculated. Similarly, the area of the signal peak for the AR gene PCR product was determined by integration and compared with the signal of the reference product peak. All reactions were conducted in triplicate, and a negative control (no template DNA) was included in each PCR test. Known unaffected control samples were also included in each set of analyses; ratios calculated from these reactions were used as reference ratios for comparison with the ratios obtained for the analyzed patient samples.
[FIGURE 1 OMITTED]
Two multiplex PCBs (PMP22 with NF1 and EW401 with NF1) were carried out in triplicate for each sample tested. Two target:reference PCR product ratios (PMP22:NF1 and EW401:NF1) were calculated from each triplicate multiplex PCR. The final target:reference PCR product ratios for PMP22:NF1 and EW401:NF1 were the average of three ratios obtained from the three replicate PCBs. Thus, two averaged ratio values were determined for each CMT1A, unaffected, and HNPP sample tested. For the known unaffected control samples, the mean PMP22:NF1 and EW401:NF1 ratio values were calculated, and each was scaled to a value of 1.0. All test sample ratios were then scaled accordingly. A single ratio (ARex4:NF1) value was used for the quantitation of the X chromosome copy number in males and females.
DETERMINATION OF THE LINEAR RANGE OF PCR
The linear range of the PCR was determined by analyzing a CMT1A, an unaffected, and an HNPP sample over 10-34 PCR cycles (data not shown), using each set of primers to be included in the quantitative analysis. Aliquots were removed, and PCR products were quantitated at 10 cycles and at every subsequent cycle thereafter, up to a total of 34 PCR cycles. Amplification products were observed after 20 cycles. However, all PCBs were in the logarithmic, or linear, phase of amplification at 26 cycles, and optimal determination and consistency of ratio values were obtained. Thus, 26 cycles was chosen as the number of cycles for quantitative analysis.
DETERMINATION OF VARIABILITY OF CALCULATED RATIOS BETWEEN GEL LANES
Variations in calculated ratio values (for the same sample) between each of the gel lanes of the detection and analysis system was assessed by comparing the relative amounts of two fragments (246 and 349 bp) of a 123-bp DNA ladder (Life Technologies). Equal amounts of the size markers were electrophoresed in each lane of the gel (data not shown), and the ratio of the quantity of the same two DNA bands was determined for each lane. Size standards were used for this analysis instead of PCR products to avoid any potential PCR-associated variability in the amplification of templates. If there is no variability, then the ratio of the quantity of the two DNA bands should be the same in every lane of the gel.
GENE DOSAGE ANALYSIS OF CMTIA, HNPP, AND UNAFFECTED INDIVIDUALS
PCR product bands detected during electrophoresis were converted to signal peaks to obtain a signal density profile (Fig. 2). The area of each of the PCR product signal peaks in Fig. 2 was calculated by integration, and the target: reference PCR product ratio for both EW401 and PMP22 amplifications was calculated for each sample analyzed. Thus, two target:reference ratio values were generated for each sample: one for analysis at the EW401 site and the second for analysis at the PMP22 site (Fig. 3). In both cases, these ratios were compared with the same reference (NF1) PCR.
For the purpose of statistical analysis, after ratios were calculated, the ratio obtained for the known unaffected individuals was normalized to a value of 1; the ratio values for each of the CMT1A and HNPP samples were scaled in relation to this value. The average of both PMP22 and EW401 ratio results for CMT1A, unaffected, and HNPP samples were 1.58 (SD = 0.18),1.02 (SD = 0.1), and 0.56 (SD = 0.09), respectively. Analysis of variance between the three sample types (CMT1A, HNPP, and unaffected) resulted in a probability value of F = 0.001 at 95% confidence limits, indicating a significant difference. Each of the two ratios alone does not completely separate the three sample types into their respective ratio groups. An average of 10% of the PMP22:NF1 ratio values and an average of 8% of the EW401:NF1 ratio values do not fall distinctly into either of the three ratio value categories. However, combining the two target:reference ratio values separated the 95% confidence limits for the three populations of samples.
[FIGURE 2 OMITTED]
ANALYSIS OF AR GENE DOSAGE IN MALES AND FEMALES
A similar analysis was carried out for the quantitation of AR gene copy number in both males and females (Fig. 4). The average target:reference ratio (ARex4:NF1) for the AR gene was 2.3 (SD = 0.11) in females and 1.1 (SD = 0.09) in males. Analysis of variance between the two groups of ratio values (females compared with males) indicated a significant difference (F <0.001). Thus, a difference of two copies of the AR gene in females compared with one copy in males was detected.
[FIGURE 3 OMITTED]
Variables that influence PCR amplification efficiency, including [Mg.sup.2+] concentration, annealing temperature, number of PCR cycles, PCR product size and composition, primer sequences, and sample DNA purity, are consistent within a single PCR; however, some variation can occur between reactions on a day-to-day basis. An indication of the amount of variation was determined by conducting multiple analyses of three unaffected individuals on 10 separate occasions. Each PCR amplification, EW401 with NF1 and PMP22 with NF1, was conducted in triplicate (on each of the 10 occasions) for each unaffected individual, and two averaged ratio values (EW401:NF1 and PMP22:NF1) were calculated. The mean of the two averaged ratios (PMP22:NF1 and EW401:NF1) obtained in each experiment was then calculated (Table 2). The results indicate negligible variation within the PCR analyses of each sample and between samples. Comparison of the ratio values obtained for each DNA sample showed a maximum SD of 0.1 for sample 2 and a corresponding CV of 1.0%. There was also no substantial variation between the three unaffected samples used in the analysis, with mean ratio values of 0.92, 1.02, and 1.04 (SD = 0.06).
[FIGURE 4 OMITTED]
VARIATION BETWEEN GEL LANES
A comparison of the ratio of the intensity of the same two fragments of a size standard, electrophoresed in each lane of the gel, was carried out. Comparison of the ratio values obtained for each lane resulted in a CV of 1%, indicating that the lane-to-lane variability across the gel is negligible. Therefore, comparison of results obtained in different lanes of the gel can be made safely. This is important because the ratios obtained for unaffected individuals are compared with those obtained for affected individuals, which are electrophoresed in different lanes of the gel.
The described quantitative PCR method has been used to detect the DNA duplication and deletion associated with two peripheral neuropathies, CMT1A and HNPP. It allowed the differentiation of the presence of one, two, and three copies of a DNA segment. The averages for both EW401 and PMP22 target:reference ratio values were 1.58, 1.02, and 0.55 for CMT1A, unaffected, and HNPP samples, respectively. An average ratio of 1.58 for CMT1A individuals is indicative of the presence of a duplication at 17p11.2 on one of the chromosomes. There are three copies of the duplicated region for every two copies of the reference region in CMT1A individuals. Similarly, HNPP individuals had an average ratio value of 0.55, indicating that there is just one copy of the target region for every two copies of the reference region corresponding to a deletion at 17p11.2. These ratio values are very close to the expected ratio values of 1.5, 1.0, and 0.5 for CMT1A, unaffected, and HNPP samples, respectively.
Before patient analyses, a multiplex PCR allowing the coamplification of a region within the potentially duplicated/deleted target site and a reference disomic gene was established. The EW401 marker had been chosen as a second target site because it also lies within the potentially duplicated or deleted target region but is telomeric to PMP22. Shorter duplicated segments that do not include the EW401 region have been described previously (20). Thus, the target primers used were positioned such that a positive duplication result for PMP22 and a negative result for EW401 would be indicative of the presence of a shortened duplication. Therefore, the choice of primers not only allowed the detection of the common (1.5 Mb) duplicated or deleted segment but also of any shortened duplicated or deleted segments if present.
The PCR is characterized by an exponential or logarithmic amplification phase that is followed by a plateau phase in which the amount of PCR product generated is no longer proportional to the initial starting template amount. Quantitative analysis is, therefore, carried out during the log phase of the reaction. In our case, 26 cycles had been determined to be the number of cycles in which all reactions were within the logarithmic phase of amplification, with a starting amount of 5 ng of DNA. Furthermore, in our experience, smaller reaction volumes (10 [micro]L) were found to enhance reproducibility and increase the efficiency of the PCR, thereby resulting in more accurate (less variable) ratio estimates.
A high degree of sensitivity is required for the detection of single copy DNA changes. The Corbett GS-2000 detection and analysis system is a laser-based detection system possessing the sensitivity required to detect small changes in PCR product amounts without lane-to-lane variability. It can also detect the change in intensity of PCR products caused by a difference of one, two, or three copies of the starting template.
This is a reliable and accurate quantitative method, as evidenced by the ratios obtained for each sample type. A further decrease in SE can be achieved by the analysis of a greater number of replicates for each sample or by the addition of a larger number of target and reference amplifications, resulting in the determination of a greater number of target:reference ratio values for each sample (21). The use of a single target: reference ratio resulted in a maximum of 10% of samples not lying distinctly within their respective target:reference ratio value group. However, when both ratio values are considered together, all samples, including those that were outliers when only a single ratio was considered, were shown to fall into their respective ratio value categories (one, two, or three copies). Calculation of two target:reference ratios (EW401: NF1 and PMP22:NF1) for each individual also led to a decrease in the dispersion of the ratio values within each group. In the cases where there was any discrepancy between the two ratio values obtained for a single sample, the particular calculated ratio was found to fall outside the confidence intervals for either CMT1A or HNPP. Any samples not lying distinctly in any of the three ratio value groups (CMT1A, unaffected, or HNPP) were reanalyzed by repeating the two multiplex PCBs and subsequent analysis five times. The mean ratio calculated from these analyses was considered the final result for these outlier samples. If, after analysis, any samples still did not fall distinctly within any of the three groups, they would need to be analyzed by other means. Such outliers could be caused by the presence of mosaicism for the duplication or deletion and could then be analyzed by fluorescence in situ hybridization (22, 23). The need to use other means of analysis has not occurred to date, because all samples analyzed gave the same result as that which had been determined previously by PFGE and Southern analysis.
In the case of X chromosome copy number determination, PCR product target:reference (ARex4:NF1) ratio values were 2.3 and 1.1 for females and males, respectively. Thus, the difference between the presence of one and two copies of the X chromosome was successfully detected. A substantial difference between the two groups was obtained with a single ratio value. This was, however, a small sample number used in the analysis and greater variation may result.
Quantitative PCR requires an internal control because the inherent inconsistency of PCR means that independent quantitation cannot be achieved. Various controls may be used for quantitative PCR, depending on whether the quantitation required is relative or absolute. Controls for absolute quantitation include: (a) an external control-in which a known quantity of DNA is amplified with the target and a standard curve generated from the reaction enables estimation of the starting amount of target DNA (24-26); or (b) an internal control-whereby the same primers are used to coamplify a known amount of a specially designed control with the target sequence (27-29). Otherwise, a relative quantitative reaction with different primers can be used to amplify a genomic template of known frequency, which is then compared with amplification reactions of the sequence of interest (26, 29-31). Amplifying a genomic template of known frequency is the control chosen for relative quantitation. Relative quantitation, as used in this instance, offers the advantage of comparing a single reference region with any number of target amplification reactions. Thus, any number of target sequences may be analyzed against the same reference amplification as long as both target and reference amplification reactions proceed with the parallel efficiency necessary for quantitative analysis.
The system described here eliminates many of the disadvantages associated with other CMT1A or HNPP diagnostic techniques, such as the requirement for radioisotopes and large amounts of DNA, and is sensitive enough to detect single-copy changes in DNA. Also, the detection system is automated, coupling the electrophoresis with the detection and analysis in a single system. The system is, therefore, suitable for the large-scale analysis of samples that is required in the diagnostic setting and has potential applications in the quantitation of the chromosomal gains and losses that occur in many tumors.
We thank Najah Nassif and John Corbett for technical advice and support and Tessy Bananis for organizational support. R. Poropat is financially supported by an Australian Postgraduate Award (Industry) and Southern Pathology (S. Andrew, Sydney, Australia).
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RENEE A. POROPAT * and GARTH A. NICHOLSON
Molecular Medicine Laboratory, University of Sydney, Concord Hospital, Concord, New South Wales 2139, Australia.
 Nonstandard abbreviations: CMT1A, Charcot-Marie-Tooth disease type 1A; HNPP, hereditary neuropathy with liability to pressure palsies; PFGE, pulsed-field gel electrophoresis; and AR, androgen receptor.
* Address correspondence to this author at: Molecular Medicine Laboratory, University of Sydney, Clinical Sciences Building, Concord Hospital, Concord, New South Wales 2139, Australia. Fax 61-2-9767 6194; e-mail email@example.com.
Received October 1, 1997; revision accepted December 10, 1997.
Table 1. PCR primer sequences and product lengths. Primer Primer sequence bp Location EW401-F 5'-GTTAGCTTGGCTTAAGCCCAGGA-3' 154 17p11.2 (D17S61) EW401-R 5'-TAGGTAGCTTTGAGGTTAGTGACC-3' NF1-F 5'-CAGGGAGATAGACAGGCAAAAAG-3' 140 17q11.2 NF1-R 5'-CCCCCAAGATAGTATTCCAGTGC-3' PMP22-F 5'-CCCTTCTGATTCCAGTTTCTCCA-3' 172 Exon 1A PMP22-R 5'-GGCGTCTTTCCAGTTTATTCAGG-3' ARex4-F 5'-GCCTCGAGTTTAGAGTCTGTGACC-3' 380 Xq11-12 (exon 4) ARex4-R 5'-ATGGATCCCCCTTATCTCATGCTCC-3' Table 2. Reproducibility of analysis of unaffected samples-target:reference PCR product ratios calculated from the average of both the EW401:NF1 and PMP22:NF1 amplifications. Unaffected Experiment no. Sample 1 (a) Sample 2 (a) Sample 3 (c) 1 0.99 0.94 1.04 2 1.04 1.07 0.95 3 0.76 0.81 0.9 4 0.93 0.97 0.98 5 1.0 1.12 1.09 6 0.98 1.18 1.06 7 0.93 1.04 1.13 8 0.87 1.03 1.08 9 0.89 1.02 1.06 10 0.8 1.0 1.12 Mean 0.92 1.02 1.04 SD 0.09 0.1 0.08 CV(%) 0.9 1.0 0.8 (a) PCR target:reference ratio values for each individual were derived from the mean of the EW401:NF1 and PMP22:NF1 ratios, each of which were conducted in triplicate in each of the 10 experiments.
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|Title Annotation:||Molecular Pathology and Genetics|
|Author:||Poropat, Renee A.; Nicholson, Garth A.|
|Date:||Apr 1, 1998|
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