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Clinical Chemistry 52:12
2203–2210 (2006)
Molecular Diagnostics
and Genetics
Quantitative Assay of Deletion or Duplication
Genotype by Capillary Electrophoresis System:
Application in Prader–Willi Syndrome and
Duchenne Muscular Dystrophy
Chia-Cheng Hung,1† Chih-Ping Chen,2, 3† Shuan-Pei Lin,3, 4 Shu-Chin Chien,5
Chien-Nan Lee,6 Wen-Fang Cheng,6 Wu-Shiun Hsieh,7 Ming S. Liu,8 Yi-Ning Su,9, 10*
and Win-Li Lin1
Background: Deletions and duplications involving
large DNA segments result in underexpression or overexpression, depending on the changes in allele dose,
and are known to cause many common disorders. Detection of allele dose variations in the human genome is
increasingly important in medical genetic diagnosis.
Methods: We used multiplex quantitative PCR coupled
with capillary electrophoresis for accurate allele dose
determination. In cases of Prader–Willi syndrome
(PWS), a total of 24 patients with PWS, as well as 205
control individuals from the general population, were
analyzed by use of multiplex quantitative PCR to amplify the FGFR2 gene, the KRIT1 gene, and the SNRPN
gene simultaneously. In cases of Duchenne muscular
dystrophy (DMD), we optimized the multiplex quantitative PCR to amplify 38 exons to analyze the DMD gene
for rapid diagnosis of 12 DMD-affected males, 12 obligate carriers from families, and 50 unaffected female
controls.
1
Institute of Biomedical Engineering, College of Medicine and College of
Engineering, and 9 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
2
Department of Obstetrics and Gynecology, 3 Department of Medical
Research, and 4 Department of Pediatrics, Mackay Memorial Hospital, Taipei,
Taiwan.
5
Departments of Medical Genetics and Obstetrics and Gynecology, China
Medical University Hospital, Taichung, Taiwan.
Departments of 6 Obstetrics and Gynecology, 7 Pediatrics, and 10 Medical
Genetics, National Taiwan University Hospital, Taipei, Taiwan.
8
Institute eGene, Inc., Irvine, CA.
† 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 protected].
Received March 31, 2006; accepted September 19, 2006.
Previously published online at DOI: 10.1373/clinchem.2006.071118
Results: We were able to unambiguously diagnose the
deletion genotype in PWS patients and identify all deletion or duplication genotypes and carrier status in DMDaffected cases with 100% sensitivity and specificity.
Conclusions: This report describes a novel single assay
that can rapidly quantify allele dose to provide accurate
clinical genetic diagnosis. This technique offers a valuable alternative for the rapid detection of genomic
deletions or duplications and decreases costs because it
does not require expensive fluorescent reagents.
© 2006 American Association for Clinical Chemistry
Deletions and duplications involving large DNA segments are known to cause many common disorders. They
lead to underexpression or overexpression, depending on
changes in allele dose (1 ). Detection of allele dose variations in the human genome is increasingly important in
medical genetic diagnosis.
Prader–Willi syndrome (PWS)11 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 12 gene on chromo-
11
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.
12
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.
2203
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Hung et al.: Quantitative Assay of Deletion or Duplication Genotype
some 15q11.2– 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 bp 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 nm
excitation light superbright light-emitting diodes based
on indium gallium nitride material technology (41 ).
Materials and Methods
patient samples
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 ␮L containing the following: 100 ng of genomic
DNA; 0.04 ␮M each primer of the FGFR2 gene; 0.08 ␮M
each primer of the KRIT1 gene; 0.2 ␮M each primer of
the SNRPN gene; 200 ␮M dNTPs; 0.5 units of AmpliTaq
Gold enzyme (PE Applied Biosystems); 2.5 ␮L of GeneAmp 10⫻ buffer II (10 mmol/L Tris-HCl, pH 8.3,
50 mmol/L KCl), in 2 mmol/L MgCl2 as provided by the
manufacturer. Amplification was performed in an MBS
thermocycler (ThermoHybaid). PCR amplification was
performed with an initial denaturation step at 95°C for
10 min, followed by 25 cycles consisting of denaturation at
94°C for 30 s, annealing at 53°C for 45 s, extension at 72°C
for 45 s, and then a final extension step at 72°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 PCRs 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 ␮L containing the
Clinical Chemistry 52, No. 12, 2006
following: 200 ng of genomic DNA; 0.04 to 0.4 ␮mol/L
each primer; 200 ␮mol/L dNTPs; 1 unit of AmpliTaq
Gold enzyme (PE Applied Biosystems); and 5 ␮L of
GeneAmp 10⫻ buffer II (10 mmol/L Tris-HCl, 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°C for 10 min
followed by 24 cycles of 94°C for 1 min, melting temperature for 1 min, 72°C for 3 min, and final extension at 72°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 ␮L of unpurified
multiplex quantitative PCR products were directly diluted 10-fold with 18 ␮L of deionized H2O. 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):
2205
Results
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 PCRs 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
Peak area of SNRPN (U)/[Peak area of FGFR2 (U)]
⫻2
Peak area of SNRPN (C)/[Peak area of FGFR2 (C)]
or
Peak area of SNRPN (U)/[Peak area of KRIT1 (U)]
⫻2.
Peak area of SNRPN (C)/[Peak area of KRIT1 (C)]
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 ).
Fig. 1. Separation of FGFR2, KRIT1, and SNRPN amplified DNA
fragments by capillary electrophoresis on the HDA system in PWS with
deletion (A), PWS without deletion (B), and unaffected individuals (C).
●, FGFR2 gene; », KRIT1 gene; ✻, SNRPN gene.
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Hung et al.: Quantitative Assay of Deletion or Duplication Genotype
Fig. 2. Total copy numbers of SNRPN gene determined by multiplex
quantitative PCR with HDA capillary electrophoresis system assay.
(A), measured for the KRIT1 gene. (B), measured for the FGFR2 gene.
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.
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. 4B) 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.
Table 1. Copy numbers of the SNRPN gene calculated by the HDA capillary electrophoresis system.
Samples
Expected gene
copy number
Measured copy number by
KRIT1 gene, mean (SD)
Measured copy number by
FGFR2 gene, mean (SD)
Unaffected individuals (n ⫽ 205)
PWS patients without deletions (n ⫽ 12)
PWS patients with deletions (n ⫽ 12)
2
2
1
2.009 (0.134)
2.028 (0.154)
1.023 (0.139)
1.992 (0.137)
2.080 (0.089)
1.046 (0.089)
Clinical Chemistry 52, No. 12, 2006
2207
Fig. 3. Exons of DMD gene detected by 8 different
multiplex quantitative PCR sets.
(A), detection by HDA capillary electrophoresis system.
(B), detection by gel electrophoresis. The DNA fragments
were separated by 1.5% agarose gel staining with
ethidium bromide.
Discussion
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 15q11– 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 15q11– 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
2208
Hung et al.: Quantitative Assay of Deletion or Duplication Genotype
Fig. 4. The results of multiplex quantitative PCR
coupled with HDA capillary electrophoresis system
analysis in affected males, carriers, and unaffected
individuals
(A), unaffected individual; (B), deleted DMD-affected male;
(C), the carrier individual with deleted exon 51; (D), duplicated DMD-affected male; (E), the carrier individual with
duplicated exon 51.
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.
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 car-
riers 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. Con-
Table 2. Various causes of PWS and comparison of results obtained with different diagnostic methods.
Percentages
60%–70%
25%–30%
⬍5%
⬍5%
Causes of PWS
Deletion in paternally derived PWS region
Maternal uniparental disomy 15 (UPD)
Chromosome translocation breaking within the PWS
critical region
Imprinting defect with variation
Multiplex
PCR
MLPA
Methylation
PCR
FISH
analysis
Sequence
analysis
Yes
No
No
Yes
Yes
No
Yes
Yes
No
Yes
No
Yes
No
No
No
No
No
No
No
Yes
2209
Clinical Chemistry 52, No. 12, 2006
versely, 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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