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Technical Briefs |
1 Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan; Departments of2 Medical Genetics, 3 Neurology,4 Pediatrics, and 5 Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan;
aaddress correspondence to this author at: Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan; fax 886-2-2392-0470, e-mail leecn{at}ha.mc.ntu.edu.tw
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) affect 1 in 3500 newborn male infants (1). DMD and BMD are both inherited in an X-linked recessive pattern resulting from mutations in the dystropin gene on Xp21.1. In approximately two-thirds of cases, the disease is inherited from female carriers, and the remaining cases come from de novo mutations in individuals without a family history of muscular dystrophy (2)(3).
Approximately 60% of DMD cases are associated with large intragenic deletions of 1 or more exons in 2 hotspot regions in the proximal and central regions of the gene (exons 3–7 and exons 44–55) (4)(5)(6)(7)(8). Approximately 6% of DMD mutations are associated with duplications of large segments (9), and the remaining cases result from point mutations, small deletions, or insertions (10)(11)(12).
Affected males can be readily detected by the absence of an amplification product in a multiplex PCR, as described by Chamberlain et al. (13) and Beggs et al. (14). Up to 98% of all frequent deletions can be detected, but in carriers, the nondeleted X chromosome hampers detection, making identification of DMD carrier status difficult. Several DMD carrier identification strategies based on quantitative Southern blotting (15), fluorescent in situ hybridization (16), linkage analysis(17), fluorescence-based strategies, microchip electrophoresis, capillary electrophoresis (18)(19)(20)(21)(22)(23)(24)(25)(26)(27), multiplex amplifiable probe hybridization (28), and multiplex ligation-dependent probe amplification (29) have been described.
Denaturing HPLC (DHPLC) is a simple, rapid, non-gel–based, non-fluorescence–based method that uses ion-pair reversed-phase liquid chromatography for detection of DNA variations; the method is sensitive and specific (30). We describe a new application combining DHPLC with multiplex PCR to efficiently and accurately identify carrier and noncarrier status in DMD families.
We analyzed 84 DNA samples from the National Taiwan University Hospital, including DNA from 11 patients with dystropin gene deletions previously detected by gel electrophoresis, 1 patient with a duplication detected by the multiplex PCR/DHPLC detection method and confirmed by quantitative real-time PCR (27), 23 obligate carriers and noncarriers from families with DMD patients, and 50 unaffected females from the general population. Genomic DNA was collected from peripheral whole blood by use of a Puregene DNA Isolation Kit (Gentra Systems) according to the manufacturer’s instructions.
To amplify the dystropin gene, multiplex PCRs of the dystropin gene were carried out as described in Chamberlain et al. (13) and Beggs et al. (14). The reaction described by Beggs et al. (14) uses multiplex I (exons 3, 50, 6, and 60) and multiplex II [muscle promoter (pm), exons 43, 13, 47, and 52]. The reaction described by Chamberlain et al. (13) uses multiplex III (exons 48, 17, 8, 44, and 46) and multiplex IV (exons 45, 19, 51, 12, and 4).
Each multiplex PCR for the specific DNA fragments was performed in a total volume of 50 µL containing 200 ng of genomic DNA, 0.04–0.4 µM each primer, 200 µM deoxynucleotide triphosphates, 1 U of AmpliTaq Gold enzyme (PE Applied Biosystems), and 5 µL of GeneAmp 10x buffer II [10 mM Tris-HCl (pH 8.3), 50 mM KCl] in 2 mM MgCl2 as provided by the manufacturer. Because amplification efficiencies differed among primer pairs in multiplex PCR, different amounts of the primers were used to obtain comparable signals within different exons. PCR amplification was performed on an MBS thermocycler (ThermoHybaid) with an initial denaturation step at 95 °C for 10 min followed by 24 cycles of denaturation at 94 °C for 1 min, annealing at 59 °C for 1 min, and extension at 72 °C for 3 min, with a final extension step at 72 °C for 10 min.
For multiplex detection, we loaded 40 µL of crude PCR product on a DNASep column (Transgenomic) and conducted DHPLC analysis in a WAVE DHPLC instrument (Transgenomic) as described previously (30). The initial and final concentrations of solvent B in the 12-min gradient were 46% and 70%, the flow rate was 0.9 mL/min, and the temperature was 50 °C.
For each sample, we measured the heights and the areas of the different peaks from each exon, using the WAVE MAKER software, which labeled the data automatically. To identify female carrier status, as indicated by the presence of large deletions or duplications in the gene dosage analysis, the copy number of a specific test exon in the unknown samples was determined by the formula:
 | (1) |
where U indicates the unknown sample and C the control sample. The reference exon is an undeleted or unduplicated exon in the same multiplex group as a deleted or duplicated/test exon.
We determined the linearity of the PCR reaction by testing the relationships among the number of PCR cycles (22–28 cycles), the DNA concentration (50–400 ng), and the amount of PCR product (31), and then standardizing the number of quantitative PCR reaction cycles to 24 cycles with a DNA concentration of 200 ng.
Using this technique to detect common exon deletions in the dystropin gene, we assigned 19 DNA fragments to 4 sets of multiplex PCR reactions according to their sizes and amplification efficiencies. To detect gene deletions, we analyzed the PCR products by both gel electrophoresis and DHPLC (Fig. 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue7/). Deleted exons are indicated by the absence of corresponding signals in the affected cases compared with controls.
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The results of multiplex PCR/DHPLC analysis of affected males, carriers, and noncarriers from different DMD families are shown in Fig. 1
. All results were analyzed by use of the ratios of deleted and undeleted exons. Every sample was analyzed at least 3 times, and the results were reproducible. Individuals were identified with decreased amplification of signals that were absent in affected individuals with deleted exons. In family C, no dosage change is apparent in the mother; therefore, de novo mutation can be deduced. In family D, more than 1 exon deletion exists in the same multiplex PCR reaction. In this situation, carrier state can still be correctly identified by calculating the ratios of tested exons to reference exons.
Measured copy numbers of the dystropin gene from carriers, noncarriers, and unaffected females, obtained by multiplex PCR/DHPLC analysis and expressed in test-exon to reference-exon ratios, are shown in Fig. 2 of the online Data Supplement. The measured copy numbers between deleted and undeleted exons can be differentiated unambiguously with the use of this analytical tool, enabling successful identification of unaffected females (Table 1 of the online Data Supplement). In our study, the peak height and peak area for PCR amplification results from each exon were divided by the reference exon, and the ratios of the unknown samples were compared with the control samples to determine the copy number of each exon. Theoretically, the copy number should be 1 for a single copy and 2 for a double copy. The mean values measured by peak height and peak area for a single and double copy are shown in Table 1 of the online Data Supplement. The values never overlapped in our controls. Large deletions in all of the DMD carriers were clearly identified with the multiplex PCR/DHPLC assay. Furthermore, 1 study patient with gene duplications was identified (Fig. 1
). Recently, a similar strategy was used to detect RB1 gene rearrangement (32).
Unlike other available techniques, including capillary gel electrophoresis, multiplex amplifiable probe hybridization, and multiplex ligation-dependent probe amplification, the multiplex PCR/DHPLC assay uses ultraviolet detection and automated instruments that can speed up carrier detection without the use of radioisotopes or fluorescence-labeled or gel-based materials. For clinical applications, a specific advantage of this detection system is the procedure for interpreting carrier status. Another advantage is that the crude PCR products do not require further purification before DHPLC analysis. Our method took 
3 h for PCR amplification and 48 min for DHPLC analysis for 4 multiplex sets. The DHPLC analysis cost was less than US $4.00 for each sample. This technique thus is well suited for routine diagnostics.
In conclusion, the multiplex PCR/DHPLC detection system is a simple, rapid, and powerful assay enabling direct detection of deletions and duplications in DMD patients and carriers. This system may also be adapted for diagnostic use in other genetic diseases involving deletion and duplication mutations.
Acknowledgments
This work was supported by grants from the National Science Council of Taiwan (NSC 93-2314-B-002-067).
Footnotes
1 C-C. Hung and Y-N. Su contributed equally to this study; 
References
The following articles in journals at HighWire Press have cited this article:
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  C.-C. Hung, C.-P. Chen, S.-P. Lin, S.-C. Chien, C.-N. Lee, W.-F. Cheng, W.-S. Hsieh, M. S. Liu, Y.-N. Su, and W.-L. Lin Quantitative Assay of Deletion or Duplication Genotype by Capillary Electrophoresis System: Application in Prader-Willi Syndrome and Duchenne Muscular Dystrophy Clin. Chem., December 1, 2006; 52(12): 2203 - 2210. [Abstract] [Full Text] [PDF]
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