Quantitative assay of deletion or duplication genotype by capillary electrophoresis system: application in Prader-Willi syndrome and Duchenne muscular dystrophy.
Prader-Willi syndrome (PWS)  is a complex developmental and neurobehavioral disorder, with an overall incidence of 1 in 10 000 newborns (2). This severe neurobehavioral disease has been reported to be caused by loss of function of paternal-derived genes from chromosome 15, especially a related SNRPN  gene on chromo some 15g11.2-q13 (3,4). Approximately 70% of PWS cases are associated with a de novo paternally derived deletion, ~25% with maternal uniparental disomy 15, and the rest with deletions or epimutations in the imprinting center or from chromosome 15q translocations (5-7).
Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders in children, affects 1 in 3500 newborn males (8) and is inherited in an X-linked recessive pattern, resulting from variations in the DMD gene on Xp21.1. The DMD gene, consisting of 79 exons and spanning a region of 2.4 million by of genomic DNA, is the largest known human gene (9,10). Approximately 55%-65% of DMD cases are associated with large intragenic deletions or duplication, ~5%-10% with duplications of large segments (11), and the remaining cases with point variations, small deletions, or insertions (12). Approximately 1/3 of cases arise from de novo variations without family history, and 2/3 are inherited from female carriers (13). Detection and identification of female carriers and noncarriers are important for genetic counseling.
In general, the hybridization-based techniques, including fluorescence in situ hybridization (FISH) (14,15) and Southern blotting (11, 16), are the most common approaches for the detection of gene deletions, but these are time-consuming and labor-intensive and cannot be used with high-throughput strategies. Moreover, Southern blotting requires relatively large amounts of genomic DNA. Multiplex amplifiable probe hybridization (17,18) and multiplex ligation-dependent probe amplification (MLPA) (19-21) are rapid techniques involving preparation of only 2 reactions and are efficient primary methods to diagnose deletion and duplication genotypes. These assays rely on detection of fluorescence, however, and require relatively expensive reagent sets.
Multiplex quantitative PCR is a new approach for the detection of gene deletions, duplications, and rearrangements (22). In recent years, several new techniques have been developed for the quantitative assay of PCR products, including fluorescence-based strategies (23-27), microchip electrophoresis (28-31), denaturing HPLC (32, 33), capillary electrophoresis (34, 35), and mass spectrometry (36). Capillary electrophoresis is a simple, high-performance, reliable, high-resolution, timesaving, and low labor-intensive technique that has shown promise as a sensitive and specific tool for the separation of biomolecules and the detection of variations in DNA (37-40).
We present a new method for high-throughput PWS deletion analysis and DMD deletion/ duplication determination by multiplex quantitative PCR coupled with a high-performance DNA analysis (HDA) system that uses capillary electrophoresis for accurate determination of allele dose. This method allows transmission of 524 run excitation light superbright light-emitting diodes based on indium gallium nitride material technology (41).
Materials and Methods
DNA samples from all PWS patients, DMD patients, family members, and unaffected individuals were obtained from National Taiwan University Hospital. A total of 229 DNA samples were analyzed in the PWS deletion study, including specimens from 24 patients with a diagnosis of PWS and 205 unaffected individuals from the general population. A total of 74 DNA samples were analyzed in the DMD deletion/ duplication study, including 12 patients with a diagnosis of DMD, 12 obligate carriers from families, and 50 unaffected females from the general population. Genomic DNA was collected from peripheral whole blood with a Chemagic DNA Blood Kit (Chemagen), according to the manufacturer's instructions. This study was approved by the Ethics Committee of the Department of Medical Genetics, National Taiwan University.
MULTIPLEX QUANTITATIVE PCR FOR PWS DELETION STUDY
Multiplex quantitative PCR was used to amplify the FGFR2, KRIT1, and SNRPN genes (FGFR2 forward, CAC AAT CAT TCC TGT GTC GT; FGFR2 reverse, AGC AGT CAA CCA AGA AAA GG; KRIT1 forward, TTC GAA TGG CTA CTT CTA CCT G; KRIT1 reverse, AAA ACG TCT TTT AAA TCA GAG C; SNRPN forward, CTT TGT ACT CCT CCA GCA AC; SNRPN reverse, TAC AGG AAT GAA AGG CAT TA). The KRIT1 and FGFR2 genes were used as internal controls for determining the relative allele dose of the SNRPN gene. All multiplex quantitative PCR amplifications were designed in a total volume of 25 [micro]L containing the following: 100 ng of genomic DNA; 0.04 [micro]M each primer of the FGFR2 gene; 0.08 [micro]M each primer of the KRIT1 gene; 0.2 [micro]M each primer of the SNRPN gene; 200 [micro]M dNTPs; 0.5 units of AmpliTaq Gold enzyme (PE Applied Biosystems); 2.5 [micro]L of GeneAmp 10x buffer II (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCI), in 2 mmol/L Mg[C1.sub.2] as provided by the manufacturer. Amplification was performed in an MBS thermocycler (ThermoHybaid). PCR amplification was performed with an initial denaturation step at 95[degrees]C for 10 min, followed by 25 cycles consisting of denaturation at 94[degrees]C for 30 s, annealing at 53[degrees]C for 45 s, extension at 72[degrees]C for 45 s, and then a final extension step at 72[degrees]C for 10 min.
MULTIPLEX QUANTITATIVE PCR FOR DMD DELETION/ DUPLICATION STUDY
Multiplex quantitative PCR of the DMD gene was performed with primers as designed in previous studies (42-44). Eight sets of multiplex PCBs were used to optimize the system and allow the amplification of 38 exons. The primers are listed in Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol52/ issue12. Each multiplex PCR for the DNA fragments was performed in a total volume of 50 [micro]L containing the following: 200 ng of genomic DNA; 0.04 to 0.4 [micro]mol/L each primer; 200 [micro]mol/L dNTPs; 1 unit of AmpliTaq Gold enzyme (PE Applied Biosystems); and 5 [micro]L of GeneAmp 10x buffer II (10 mmol/L Tris-HCI, pH 8.3, 50 mmol/L KCl) in 2 mmol/L MgCl2 as provided by the manufacturer. PCR amplification was performed on an MBS thermocycler (ThermoHybaid) with 95[degrees]C for 10 min followed by 24 cycles of 94[degrees]C for 1 min, melting temperature for 1 min, 72[degrees]C for 3 min, and final extension at 72[degrees]C for 10 min.
HDA CAPILLARY ELECTROPHORESIS SYSTEM
For rapid DNA separation and detection we used an HDA capillary electrophoresis system with CK-5000 disposable cartridge (eGene) (41). The gel-matrix in the gel cartridge consists of proprietary linear polymer with ethidium bromide dye. The system was used according to the manufacturer's operation manual: 2 [micro]L of unpurified multiplex quantitative PCR products were directly diluted 10-fold with 18 [micro]L of deionized HZO. The samples were placed in the instrument sample tray and were automatically injected into the capillary channel and subjected to electrophoresis by selecting the OM900.mtd method from BioCalculator software. The sample injection voltage was 5 kV with sample injection time of 20 s followed by separation voltage of 3 kV and separation time of 900 s. The system can simultaneously analyze 12 PCRs in 10 min.
DETERMINATION OF TOTAL SNRPN GENE COPY NUMBER
The specific multiplex quantification PCR was analyzed by the HDA capillary electrophoresis system. The quantification of DNA fragments was based on the integrated peak area automatically determined by BioCalculator software. Total SNRPN gene copy numbers were calculated by adjusting the relative known doses of the FGFR2 and KRIT1 genes. We used the following equation to calculate the copy number of the SNRPN gene in the unknown samples (U) compared with the control samples (C):
Peak area of SNRPN (U)/[Peak area of FGFR2 (U)]/x Peak area of SNRPN (C)/[Peak area of FGFR2 (C)] 2
Peak area of SNRPN (U)/[Peak area of KRIT1 (U)]/x Peak area of SNRPN (C)/[Peak area of KRIT1 (C)] 2.
DETERMINATION OF DMD GENE COPY NUMBER
In the DMD deletion/ duplication study, we used BioCalculator software to measure the areas of the different peaks from each exon. To identify carrier status in females on the basis of findings of large deletions or duplications in the allele dose analysis, we determined the copy number of specific test exons in the unknown samples as described in our previous report (33).
MULTIPLEX QUANTITATIVE PCR
We performed multiplex quantitative PCR amplification with a total of 229 DNA samples to identify the PWS genotype with the SNRPN gene segment and the control FGFR2 and KRIT1 gene segments. For the DMD deletion/ duplication study, the technique was also used to detect common exon deletions and duplications in the DMD gene. Thirty-eight DNA fragments were assigned to 8 sets of multiplex quantitative PCBs according to their sizes. Annealing temperature, template concentration, primer concentrations, and number of cycles were optimized for different genes/ amplicons. We tested the relationship between the number of PCR cycles and the amount of PCR product to determine the linearity of the PCR (45). Up to 25 cycles, PCR is in the exponential phase (46, 47). Therefore, we standardized the number of PCR cycles in our multiplex quantitative PCR to 25 cycles.
CAPILLARY ELECTROPHORESIS FOR PWS DELETION STUDY
The PCR products had different fragment sizes: FGFR2, 251 bp; KRIT1, 326 bp; and SNRPN, 448 bp. The diluted crude PCR products were injected directly into the capillary channel on the HDA system. The results of analysis of amplification by the HDA system of FGFR2, KRIT1, and SNRPN gene segments in several samples from PWS patients with deletions, PWS patients without deletions, and unaffected individuals are shown in Fig. 1. Deletions in the PWS patients were further confirmed by FISH studies with SNRPN probes (see Fig. 1 in the online Data Supplement). Diagnostic results obtained in PWS patients with deletion by the HDA capillary electrophoresis system were comparable to those obtained with FISH technology.
QUANTIFICATION OF SNRPN GENES
The 3 continuous peaks shown in Fig. 1 represent the FGFR2, KRIT1, and SNRPN genes, respectively. Total copy numbers of the SNRPN gene calculated from the results of multiplex quantitative PCR for the reference gene (KRIT1) in 24 PWS patients and 205 unaffected individuals from the general population are graphically displayed in Fig. 2A. The results of calculation of the copy numbers with FGFR2 as the control gene are shown in Fig. 2B. Comparison of results revealed patients had similar copy numbers for these genes.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
The SNRPN gene copy number in samples from PWS patients with deletions was virtually identical to the expected value of 1, and the nondeletional samples and those from unaffected populations were close to a copy number of 2. Statistical analysis revealed that deletion type could be distinguished from nondeletion type with an uncertainty of P <0.0001 (Table 1). To test the validity and reproducibility of our system for dose determination of the SNRPN gene, we analyzed patient samples repeatedly at least 3 times, and the results were all demonstrated to be reproducible.
CAPILLARY ELECTROPHORESIS FOR DMD DELETION DUPLICATION STUDY
After amplification of the DMD gene, we analyzed the multiplex PCR products with both gel electrophoresis and the HDA capillary electrophoresis system for the detection of different exons of the DMD gene (Fig. 3A). The corresponding signals in the HDA capillary electrophoresis system were compared with bands separated by gel electrophoresis and stained with ethidium bromide (Fig. 3B). The lower sensitivity of ultraviolet gel-based detection led to differences between band intensities on gel and values obtained with the HDA method.
The results of multiplex quantitative PCR coupled with HDA capillary electrophoresis system analysis in affected males, carriers, and unaffected individuals are shown in Fig. 4. The results were analyzed by use of the ratios of deleted and undeleted exons. The absence of corresponding signals in the affected cases compared with unaffected controls (Fig. 413) indicates the deleted exons in the DMD patient. The decreased amplification in the corresponding signals for deleted exons in a DMD carrier are shown in Fig. 4C, and increased amplification in the corresponding signals in a DMD patient with duplicated exons and a carrier are shown in Fig. 4, D and E. Every sample was analyzed at least 3 times, and the results were reproducible.
QUANTIFICATION OF DYSTROPIN GENES
The copy numbers of the DMD gene as determined by multiplex quantitative PCR and HDA capillary electrophoresis system analysis in deleted carriers, nondeleted carriers, and unaffected females expressed in test exon: reference exon ratios are shown in Fig. 2 in the online Data Supplement. Use of this analytical tool with the measured copy numbers allowed unambiguous differentiation between deleted and undeleted exons, enabling successful identification of unaffected females and deleted carriers. Moreover, we successfully determined that 1 of the DMD patients had duplication of exons 13-17 and another had duplication of exons 50-58. There was no diagnostic value within the indeterminate interval, even for females with unknown status (see Table 2 in the online Data Supplement).
QUANTITATIVE REAL-TIME PCR
We used a quantitative real-time PCR assay to verify the status of the deleted carriers and nondeleted carriers identified by the multiplex quantitative PCR with HDA capillary electrophoresis system analysis (48). Results of allele dose analysis for exons 6 and 51 according to their deletion rates in the DMD patients are shown in Fig. 3 in the online Data Supplement. When the DNA sample was from a carrier of a deletion including exon 6 or 51, the signal corresponding to the deleted exon was approximately half that of an unaffected female [1.12 (0.18) for exon 6 and 1.15 (0.20) for exon 51]. The findings of the quantitative real-time PCR-based assay were compatible with those of multiplex quantitative PCR and HDA capillary electrophoresis system allele dose analysis for both deleted carriers and nondeleted carriers. These findings demonstrated that this multiplex quantitative PCR technique can reliably detect DMD-affected males and female carriers.
[FIGURE 3 OMITTED]
HDA is a high-throughput, multichannel, and microcapillary electrophoresis method that provides a simple, rapid, and reliable alternative to genetic quantification. This method allowed the precise and efficient identification of trisomic and disomic animals (49). In designing the multiplex PCR used in the present study, we chose to amplify the diagnostic SNRPN gene as the largest product rather than as a size between the 2 internal standard genes. Variations in the quality of the extracted DNA can have major effects on amplification efficiency, with the largest effects seen in the larger products in a multiplex reaction. However, our previous multiplex quantitative genotype analysis studies with HPLC instruments showed that competitive PCR allowed the accurate determination of allele dose variations (32, 33, 50, 51).
PWS is a complex genetic disorder characterized by small hands and feet, short stature, poor sucking, feeding difficulties, hypotonia, hypogonadism, hyperphagia, early childhood obesity, and a chromosome 15811-q13 deletion in the majority of cases. The current diagnostic approach when the syndrome is suspected is methylation testing and FISH with the SNRPN and control probes from chromosome 15. Methylation PCR is accurate in 99% of PWS patients, and 70% of PWS patients have the chromosome 15811-q13 deletion confirmed with FISH analysis and methylation PCR. In cases of FISH results indicating the wild-type gene but continued suspicion of PWS, analysis of DNA obtained from parents and the affected individual should be performed to detect possible maternal disomy 15 and/or an imprinting defect. In this report, we describe an alternative molecular method that uses capillary electrophoresis based quantitative assay for determining the deletion status. This assay is less expensive and less time-consuming than FISH analysis, but for identifying the typical deletion seen in PWS patients it is as accurate as chromosome testing and the SNRPN probe. The results obtained with this multiplex PCR/HDA system are compared with findings by other methods to diagnose PWS in Table 2. To calculate the total SNRPN allele dose, we used 2 autosomal genes outside of chromosome 15 (KRIT1 and FGFR2) as controls with a copy number of 2 in relation to the SNRPN probe, which would have a copy number of only 1 in a PWS patient with a paternally derived deletion.
[FIGURE 4 OMITTED]
DMD is the most common lethal neuromuscular genetic disorder in males. Affected patients usually present with proximal muscle weakness and pseudohypertrophy at an early age, invariably become wheelchair bound, and eventually die prematurely. Effective treatment has not been found, and current management strategies consist of identifying DMD carriers so that early interventions can be carried out. Because DMD follows an X-linked inheritance pattern, female relatives of DMD patients are at risk of being carriers. To institute early interventions such as genetic counseling and prenatal diagnosis, a fast and cost-effective test that can accurately identify at-risk carriers is crucial. In the past, increased serum creatine kinase was used to identify asymptomatic carriers (52). Under many circumstances, however, creatine kinase values may be within the reference interval in approximately 1/3 of female carriers. Therefore, carrier detection by DNA analysis is still the most accurate technique. To detect the DMD gene deletion/ duplication in affected males and carriers in an efficient way, we devised an HDA capillary electrophoresis system for quantitative PCR-based assay. The strategy is based on quantitative multiplex PCR of the deleted /duplicated and undeleted/ unduplicated exons of the patient and the obligate carrier in the family. In patients with DMD, the deleted/ duplicated exons can be easily identified by direct visual inspection of the absence/ increase of the corresponding peak. For carrier detection, the carrier status can be confirmed by calculating the alteration in the allele dose by use of the deleted/ duplicated to undeleted/unduplicated exon ratio. In the present study, the value of the peak area for PCR amplification from each exon was divided by that of the reference exon, and the ratios of the unknown samples were compared with the control samples to determine the copy number of each exon. When the value of the copy number from the sample was higher than 1.5, the result was classified as unaffected. Conversely, if the value was <1.5, the result was classified as deleted carrier. No overlap was observed between the values obtained for single and double copy number.
This novel multiplex quantitative PCR method for deletion or duplication analysis was easier and faster than traditional diagnostic approaches. In addition, the HDA system based on capillary electrophoresis was suitable for analyzing large numbers of samples and accommodated high sample throughput. Furthermore, the diluted crude PCR products are directly and automatically injected into the capillary channel without the need for further purification or denaturation. This is not a turn-key system, however, and users must be trained to accurately interpret the results.
This analytic method can efficiently analyze 12 PCR products simultaneously in 10 min. For DMD diagnosis, the approximately $32 per patient cost of 2 different probe mixes of MLPA reagent sets is a significant decrease compared with preexisting techniques. The MLPA process requires the use of an expensive fluorescent-based sequencer/DNA analyzer such as an ABI 3730/ABI 3100, whereas the proposed process uses a much less expensive eGENE HDA capillary electrophoresis system. The cost of a single multiplex assay is about $2.50, and it costs approximately $20 per patient to perform 8 sets of multiplex PCR coupled with use of the HDA capillary electrophoresis system. Compared with quantification assay labeled with fluorescent dye, our method is a simple, inexpensive, and in-house protocol with a reliable quantification method well suited for measuring gene copy numbers.
In conclusion, we report a powerful, rapid, and extremely reliable quantitative PCR assay and demonstrate its clinical application for the detection of deletion or duplication genotypes. This assay can be used for quantification of the SNRPN gene copy numbers in deletion/nondeletion types of PWS, as well as for identification of deletion/ duplication genotype in affected male and female carriers for diagnosis of DMD. This study showed that the sensitivity and specificity of this multiplex quantitative PCR coupled with HDA capillary electrophoresis analysis are consistent with those of conventional FISH and real-time PCR analysis. This attractive alternative method is a promising tool for the detection of disorders involving deletions and duplications, such as PWS, Angelman syndrome, and DiGeorge syndrome.
We are very grateful to all the families who participated in this research. We acknowledge Dr. Su-Ming Hsu for English language editing and Dr. Fon-Jou Hsieh for expert assistance. This work was supported by the National Science Council of Taiwan (NSC 93-2314-B-002-174; NSC 94-3114-P-002-002-Y5).
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CHIA-CHENG HUNG, ([dagger]) CHIH-PING CHEN [2,3]([dagger]) SHUAN-PEI LIN [3,4] SHU-CHIN CHIEN,  CHIEN-NAN LEE,  WEN-FANG CHENG,  WU-SHIUN HSIEH,  MING S. LIU,  YI-NING SU, [9,10] * and WIN-LI LIN 
 Institute of Biomedical Engineering, College of Medicine and College of Engineering, and  Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.  Department of Obstetrics and Gynecology,  Department of Medical Research, and  Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan.  Departments of Medical Genetics and Obstetrics and Gynecology, China Medical University Hospital, Taichung, Taiwan.  Institute eGene, Inc., Irvine, CA.  Nonstandard abbreviations: PWS, Prader-Willi syndrome; DMD, Duchenne muscular dystrophy; FISH, fluorescence in situ hybridization; MLPA, multiplex ligation-dependent probe amplification; HDA, high-performance DNA analysis.  Human genes: SNRPN, small nuclear ribonucleoprotein polypeptide N; DMD, dystrophin (muscular dystrophy, Duchenne and Becker types); FGFR2, fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome); KRIT1, KRIT1, ankyrin repeat containing.
Departments of  Obstetrics and Gynecology,  Pediatrics, and  Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
([dagger]) These authors contributed equally to this study.
* Address correspondence to this author at: Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
Fax 886-2-23813690; e-mail: email@example.com.
Received March 31, 2006; accepted September 19, 2006.
Previously published online at DOI: 10.1373/clinchem.2006.071118
Table 1. Copy numbers of the SNRPN gene calculated by the HDA capillary electrophoresis system. Expected Measured copy gene copy number by KRIT1 Samples number gene, mean (SD) Unaffected individuals (n = 205) 2 2.009 (0.134) PWS patients without deletions (n = 12) 2 2.028 (0.154) PWS patients with deletions (n = 12) 1 1.023 (0.139) Measured copy number by FGFR2 Samples gene, mean (SD) Unaffected individuals (n = 205) 1.992 (0.137) PWS patients without deletions (n = 12) 2.080 (0.089) PWS patients with deletions (n = 12) 1.046 (0.089) Table 2. Various causes of PWS and comparison of results obtained with different diagnostic methods. Multiplex Percentages Causes of PWS PCR 60%-70% Deletion in paternally derived PWS region Yes 25%-30% Maternal uniparental disomy 15 (UPD) No <5% Chromosome translocation breaking within No the PWS critical region <5% Imprinting defect with variation No Methylation FISH Sequence Percentages MLPA PCR analysis analysis 60%-70% Yes Yes Yes No 25%-30% Yes Yes No No <5% No No Yes No <5% No No No Yes
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Hung, Chia-Cheng; Chen, Chih-Ping; Lin, Shuan-Pei; Chien, Shu-Chin; Lee, Chien-Nan; Cheng, Wen-Fang;|
|Date:||Dec 1, 2006|
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