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Technical Briefs |
1
Department of Chemistry, McCormick Road, University of Virginia, Charlottesville, VA 22901
2
Molecular Genetics Laboratory, Mayo Clinic, Rochester, MN 55905
3
Department of Pathology, University of Virginia Health Science Center, Charlottesville, VA 22901
aauthor for correspondence: fax 804-982-3048, e-mail jpf3p{at}virginia.edu
Duchenne muscular dystrophy (DMD), a genetic disease caused by mutations in the X chromosome, is currently diagnosed by Southern blot analysis. Affected males and female carriers are identified by the detection of duplicated or deleted exons in the dystropin gene. Disadvantages of this method include the large number of fragments required for hybridization to determine genomic alterations and the time-consuming, expensive, and tedious nature of the analysis. The deletions/duplications seen in DMD tend to be located at certain "hot spot" regions of the gene, however, allowing easy detection of a great number of cases by interrogating a limited number of PCR-amplified DNA fragments.
Chamberlain and coworkers (1)(2) developed primers to amplify nine fragments from known deletion sites in a single multiplex PCR amplification. Beggs et al. (3) defined additional primers that amplify nine fragments in a second multiplex PCR. This total set of 18 fragments allows >97% of the deletions detectable by Southern blot analysis to be detected rapidly and accurately by PCR.
Analysis of the PCR fragments is most frequently carried out on slab gels, but both the presence and amount of the correctly sized fragments are important because carriers have both the mutated and wild-type gene. Many clinical laboratories that offer DMD testing do not provide carrier testing because of the technical difficulties associated with quantitative analysis. To speed analysis and automate quantification, several groups have exploited capillary electrophoresis (CE) for the separation of DMD-relevant fragments (4)(5)(6). One of the groups that reported DMD detection by CE transferred their method to an electrophoretic microdevice (6), a very rapid separation technique demonstrated to be an effective tool for use in clinical analyses (7)(8)(9)(10)(11)(12)(13). Although these reports show CE and microchip electrophoresis as a viable and much more rapid method for DMD detection, analyses were performed under different conditions, with limited patient samples; thus these methods are not likely to be transferred to the clinical laboratory.
Recently, a commercial microchip electrophoresis instrument has been introduced that relies on standardized protocols and reagents for consistent results (14). This work is a primary evaluation of this microchip electrophoresis instrument for diagnosis of DMD in both affected males and carrier females. Clinical application was tested by analyzing 50 patient samples and comparing the mutation diagnoses from this method with those from the conventional method (Southern blot).
Traditional Southern blot analysis was performed by isolating genomic DNA from whole blood using a Puregene reagent set (Gentra Inc). The DNA, digested with BglII and HindIII, was electrophoresed through 0.8% agarose and transferred by Southern blot to a nylon membrane. The membrane was prehybridized for 2–6 h in formamide hybridization buffer; [32P]dCTP-labeled probe was then added to give 0.2–1.0 cpm/mL (15). Probes used were cDNAs 1-2A, 2B-3, 4-5A, 5B-7, 8, and 9 (American Type Culture Collection), radiolabeled by use of the High Prime reagent set (Roche Molecular Biochemicals). Membranes were hybridized overnight at 42 °C, and then washed with 0.2x standard saline citrate at 60 °C. Results were obtained by use of a phosphorimager, and densitometry was performed using ImageQuant software (Molecular Dynamics). For data analysis, peak areas within each lane were normalized against one of the peak-area values in that same lane. The presence of a deletion or duplication in patient samples was determined by comparison of normalized values to those for non-DMD male and non-DMD female control samples.
The same genomic DNA samples were PCR-amplified in two multiplex reactions using subsets of the available primers. The Chamberlain multiplex reaction, containing primers for exons 8, 17, 19, 44, 45, and 48, was carried out for 30 cycles (94 °C for 30 s to denature, 55 °C for 30 s to anneal, and 68 °C for 2 min to extend). The Kunkel multiplex reaction, containing primers for exons 3, 6, 13, 43, 47, 60, and the promoter region, ran for 25 cycles (94 °C for 30 s to denature and 65 °C for 2 min to anneal and extend). Each multiplex PCR also included primers for a 316-bp control fragment from the ß-globin gene. Microchip electrophoresis of the PCR multiplex reactions was carried out in an Agilent BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA) using commercially available DNA 500 assay sizing reagent sets. Each multiplex mixture was diluted 1:1 with deionized water before being added to the internal marker/buffer solution in the sample wells on the microchip.
Shown in Fig. 1
are electropherograms from analysis of three samples by microchip electrophoresis: a non-DMD control (Patient 20), an affected male (Patient 21), and a female carrier (Patient 22). Visual inspection easily detected the duplication in exon 6 of the affected male and the deletion of exon 47 from the female carrier. The deletion of exon 45, diagnosed by the Southern blot method, in the female carrier was harder to detect by simple visual inspection; this is not unexpected because mutations in female carriers are often difficult to detect. A numeric method for evaluation of DMD mutation diagnoses, using the peak areas automatically calculated by the microchip instrument, might allow better comparisons with the densitometry diagnoses.
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The numeric method for evaluation of DMD mutation diagnoses was attempted with the current samples, recognizing that the PCR reactions that were carried out were not truly quantitative because of the number of cycles performed. As PCR proceeds, the exponential amplification stops once the concentration of a fragment exceeds a certain value (16). For multiplex samples, in which each fragment is amplified at a different rate, terminating the reaction before any of the fragments reaches its inherent limit is critical if quantification is required. Yau et al. (17), using fluorescently labeled primers to amplify 25 exons of the dystropin gene in two multiplex reactions, found it necessary to reduce the number of PCR cycles to less than 20 to obtain accurate quantification. Use of the current samples, however, allowed us to develop and, in a preliminary manner, evaluate a possible numeric method for DMD mutation detection.
It is well known with PCR that, even with limiting the number of cycles and using multiplex PCR primer sets designed to have approximately the same annealing temperature, not all of the DNA targets amplify to the same extent. Moreover, variations in the concentration of amplified product between PCR amplification reactions are even more common. Consequently, a normalization procedure similar to that used for normalization of densitometry data was used with the peak-area results. Reference values for each exon were established using samples from 15 non-DMD control patients that had been included in the 50 samples analyzed. As expected with the number of cycles used in the amplifications, the reference values showed a great deal of variability (Table 1
). Also given in Table 1
are the normalized concentration data from the electropherograms shown in Fig. 1
. Although the deletion of exon 45 in the female carrier was hard to distinguish by visual inspection, the normalized concentration of this exon clearly falls outside the reference values, indicating a deletion of this exon.
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For the 35 patient samples evaluated, all 35 had exon concentrations that fell outside the reference values. This indicates that, even with less than ideal amplifications and the semiquantitative nature of the protocol, DMD could be diagnosed by microchip electrophoretic analysis. The results were also encouraging for defining the nature of the mutations present. The mutations in 11 of 12 affected males were correctly identified when compared with the Southern blot results. In female carriers, mutations in 16 of 23 patients were correctly determined, with all misdiagnoses attributable to the high variability in the reference values. This indicates that more stringent PCR conditions have the potential to greatly improve the ability of this technique to correctly identify mutations in both affected males and female carriers.
The true value of the microchip separation method, however, can be appreciated by the speed of analysis alone. The analysis of both multiplex PCR samples from all 50 patients was accomplished in <5 h. This included 
15 min for preparation and loading of each microchip, with a total "hands-on" time of slightly more than 2 h. All 100 separations took less time than it would have taken to perform a single electrophoretic slab gel separation with only 10–20 PCR samples being analyzed. When the conventional Southern blot procedure is included, an even more substantial time savings is evident. This mirrors the results obtained in an earlier study comparing the detection of hepatitis C and herpes simplex viral infections via agarose gel electrophoresis–Southern blot analysis and CE (18).
The development of new methods and platforms for clinical analysis that can easily be transferred to the clinical laboratory is an important effort. This study indicates that the microchip electrophoresis method for DMD mutation detection has many advantages over the current Southern blot method. Most important are the substantial savings in time, labor, and reagent costs, with the elimination of radioactive reagents being an additional benefit. Direct transfer of the microchip method to the clinical laboratory setting is easily possible because it uses commercially available instrumentation, with standardized reagents and protocols. The preliminary evaluation carried out here shows the potential of this method for DMD detection, but also indicates that issues relating to the use of PCR amplification as a quantitative procedure in clinical laboratories remain.
Acknowledgments
We wish to thank Tammy Price-Troska (Molecular Genetics Laboratory, Mayo Clinic, Rochester, MN) for her work in preparing the PCR-amplified samples. We also wish to thank Agilent Technologies for the BioAnalyzer 2100 instrument and the DNA 500 assay reagent sets used in this study.
References
The following articles in journals at HighWire Press have cited this article:
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  S. F.Y. Li and L. J. Kricka Clinical Analysis by Microchip Capillary Electrophoresis Clin. Chem., January 1, 2006; 52(1): 37 - 45. [Abstract] [Full Text] [PDF]
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C.-C. Hung, Y.-N. Su, C.-Y. Lin, C.-C. Yang, W.-T. Lee, S.-C. Chien, W.-L. Lin, and C.-N. Lee Denaturing HPLC Coupled with Multiplex PCR for Rapid Detection of Large Deletions in Duchenne Muscular Dystrophy Carriers Clin. Chem., July 1, 2005; 51(7): 1252 - 1256. [Full Text] [PDF]
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