Clinical Chemistry 51, No. 11, 2005
cells were 0.750 and 0.914, respectively. A linear correlation
also existed between tumor mass and the plasma concentrations of human albumin gene (r2 ⫽ 0.746 and 0.625 for C666and Xeno-2117–inoculated mice, respectively). In the 4 nude
mice inoculated with HK1 cells, an EBV-negative cell line,
the human albumin gene, human mitochondrial ND6 gene,
and murine ␤-globin gene sequences, but not EBV DNA
sequences, were detected in the plasma. The plasma human
albumin gene concentrations showed a linear relationship
with the tumor mass (r2 ⫽ 0.808). Because the only source of
EBV DNA and human DNA in the mouse model was the
engrafted tumor, the plasma concentrations of EBV DNA
and human albumin gene would represent the concentrations of tumor-derived nuclear DNA. Our findings therefore
suggest a direct linear relationship between the plasma
concentrations of tumor-derived nuclear DNA and the tumor mass.
On the other hand, we found no linear correlation
between the tumor mass and the human mitochondrial
ND6 gene (r2 ⫽ 0.123 and 0.374) or the murine ␤-globin
gene (r2 ⫽ 0.363 and 0.037) concentrations in mice inoculated with C666 and Xeno-2117 tumor cells. We measured
the human mitochondrial ND6 gene concentrations in the
resected tumor tissues and found that there was a wide
variation of up to 5-fold even within the same type of
cancer tissues (data not shown). One explanation would
be the variable number of mitochondria in each tumor cell
and the variable number of mitochondrial DNA genomes
in each mitochondrion (19 ). In the 5 mice not inoculated
with tumor cells, only the murine ␤-globin gene was
detected in the plasma.
In summary, our results substantiate the tumoral origin
of circulating EBV DNA in NPC patients and show that
the concentrations of EBV DNA and other tumor-derived
nuclear DNA sequences vary in a linear fashion with the
mass of the NPC.
The section of the text concerning plasma EBV genotyping
was supported by a grant from the Research Grants
Council of the Hong Kong Special Administrative Region,
China (Project CUHK 4086/02M), and the section correlating tumor mass and plasma EBV DNA concentrations
was supported by the Kadoorie Charitable Foundations
(under the auspices of the Michael Kadoorie Cancer
Genetics Research Program).
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2. Mutirangura A, Pornthanakasem W, Theamboonlers A, Sriuranpong V,
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DOI: 10.1373/clinchem.2005.054783
Reliable and Cost-Effective Screening of Inherited
Heterozygosity by Zn2ⴙ–Cyclen Polyacrylamide Gel
Electrophoresis, Eiji Kinoshita,1,2* Emiko Kinoshita-Kikuta,1
Hirokazu Kojima,1 Yukiko Nakano,3 Kazuaki Chayama,3
and Tohru Koike1,2 (1 Department of Functional Molecular
Science, Division of Medicinal Chemistry, Graduate
School of Biomedical Sciences, 2 Frontier Center for Microbiology, and 3 Department of Medicine and Molecular
Science, Division of Frontier Medical Science, Graduate
School of Biomedical Sciences, Hiroshima University,
Hiroshima, Japan; * address correspondence to this author at: Department of Functional Molecular Science,
Division of Medicinal Chemistry, Graduate School of
Biomedical Sciences, Hiroshima University, Hiroshima
734-8551, Japan; fax 81-82-257-5336, e-mail kinoeiji@
hiroshima-u.ac.jp)
Various procedures have been developed (1– 4 ) to facilitate high-throughput single-nucleotide polymorphism
2196
Technical Briefs
(SNP) detection for establishment of genetic linkage and
aiding in diagnosis and treatment of inherited diseases.
Such procedures, however, usually require expensive
equipment and skilled analysts, making it very difficult
for most clinical researchers and physicians to obtain
useful SNP data. Thus, the establishment of a reliable and
cost-effective SNP detection method that uses general
equipment is desirable.
We developed a simple, rapid, cost-effective, and
accurate method for the detection of mutations by polyacrylamide gel electrophoresis with the additive Zn2⫹–
1,4,7,10-tetraazacyclododecane (cyclen) complex (Rf Enhancer-ZC; Toyobo), called Zn2⫹– cyclen–PAGE (5 ). The
combination of a PCR-based heteroduplex method and
Zn2⫹– cyclen–PAGE enables accurate detection of single
mutations introduced artificially, even for less detectable
substitutions such as A-T to T-A and G-C to C-G. This
procedure does not require radioisotopes or fluorescent
probes. Heteroduplex bands that arise from annealing of
complementary strands, one from mutant and one from
wild-type DNA (heterozygosity) are identified in the
electrophoresis gel during PCR.
Our approach is based on 3 principles: (a) a single-base
mismatch produces a local conformational change in the
double-stranded DNA, leading to differential migration
of the heteroduplex and homoduplex bands; (b) the addition to the gel of Zn2⫹– cyclen, which selectively binds to
the thymine base (T) and disrupts the double strands,
intensifies the local conformational change, increasing
differential migration of both duplexes; and (c) binding of
Zn2⫹– cyclen to T decreases the total charge of the target
DNA, thus enhancing detection. Slow or differentially
migrating bands in the gel indicate the presence of heteroduplex bands, which suggest the existence of a mutation or polymorphism. Furthermore, Zn2⫹– cyclen–PAGE
separates homoduplexes of specific mutant alleles from
homoduplexes of their homologous wild-type alleles. We
achieved higher resolution of Zn2⫹– cyclen–PAGE by
adopting a discontinuous buffer system with a separating
gel and a stacking gel (6 – 8 ).
As the first practical use of the improved method, we
analyzed heterozygosity in the human cardiac sodium
channel gene, SCN5A. Many mutations in the SCN5A
gene, which consists of 28 exons spanning ⬃80 kb on
chromosome 3, are responsible for multiple arrhythmia
disorders, including long QT syndrome type 3 (LQT3),
idiopathic ventricular fibrillation (IVF), inherited cardiac
conduction defects, and the Brugada syndrome. More
than 80 mutations associated with the Brugada syndrome
have been identified (9 ) since the first indication of a
genetic basis in 1998 (10 ). Because these mutations are
scattered throughout the SCN5A gene, a comprehensive
and accurate genomic analysis is required to confirm the
classification of Brugada syndrome as an inherited disease, to predict the clinical phenotype, and to develop
suitable therapies.
Eighteen unrelated Japanese individuals participated in
this study: 10 patients with Brugada syndrome, 1 with IVF
without ST segment elevation in the right precordial
electrocardiogram leads, 1 with LQT3, and 6 healthy
individuals without family histories of syncope or sudden
death. All disease diagnosis was performed at Hiroshima
University Hospital. The study protocol was approved by
the human genome research ethics screening committee
of Hiroshima University, and written informed consent
for participation was obtained from all participants. Peripheral blood (10 mL) was obtained from each participant, and genomic DNA was extracted from the leukocytes according to a standard protocol by use of the
QIAamp DNA Blood Maxi Kit (Qiagen). All SCN5A exons
and those splice sites were amplified by PCR from 2.5 ng
of genomic DNA with KOD-plus or Blend Taq-plus DNA
polymerase (Toyobo). The untranslated region of exon 28
was not analyzed in this study. The PCR procedure was
described previously (11 ). The optimum primers used (98
pairs; primer sets are listed in the Data Supplement that
accompanies the online version of this Technical Brief at
http://www.clinchem.org/content/vol51/issue11) were
originally designed according to the sequences of GenBank accession nos. AP006241 (chromosome 3) and
M77235 (cDNA of SCN5A) so that, for accurate mutation
detection, the length of each PCR product would not
exceed ⬃200 bp. The primers were purchased from Texas
Genomics Japan and Espec Oligo Service.
The modified Zn2⫹– cyclen–PAGE was performed at
25 mA for 100 min at room temperature in a 1-mm-thick,
9-cm-wide, and 9-cm long gel on a standard minislab
PAGE apparatus (Model AE6500; ATTO). The gel consisted of 1.8 mL of stacking gel (45 g/L polyacrylamide
and 125 mmol/L Tris-HCl, pH 6.8) and 6.3 mL of a
separating gel (5.0 mmol/L Zn2⫹– cyclen, 200 g/L polyacrylamide, and 375 mmol/L Tris-HCl, pH 8.8). The
acrylamide stock solution was prepared as a mixture of a
99:1 ratio of acrylamide to N,N⬘-methylenebisacrylamide.
The cathode buffer was 25 mmol/L Tris and 192 mmol/L
glycine, and the anode buffer was 25 mmol/L Tris and
192 mmol/L glycine containing 5.0 mmol/L Zn(NO3)2.
Each PCR product for Zn2⫹– cyclen–PAGE was dissolved
in a half amount of a loading dye containing 50 mmol/L
EDTA, 0.5 g/L bromphenol blue, and 300 mL/L glycerol
and then applied (0.5–1.0 ng of DNA per well). The DNA
bands were visualized by staining with 10 000-fold– diluted SYBR Green I (15 mL per gel; Cambrex Bio Science
Rockland) after electrophoresis. The entire amount of
SCN5A gene (i.e., 41 DNA fragments ⫻ 12 patients) and
the DNA fragments, including the samples showing multiple bands (healthy individuals nos. 14 and 16), were
sequenced with an ABI PRISM 310 Genetic Analyzer
(Applied Biosystems). The primers (41 pairs) used for
direct sequencing were the same as those reported previously (12 ). Direct sequencing revealed that all patients
had the SCN5A coding the same hH1c channel isoform
(13 ), entered as GenBank accession no. AY148488.
This novel screening method disclosed that 9 patients
and 2 healthy individuals had various heterozygosities in
the SCN5A gene. Examples of typical Zn2⫹– cyclen–PAGE
results are shown in Fig. 1. The higher resolution achieved
with the same PCR products from exon 10 for electro-
Clinical Chemistry 51, No. 11, 2005
Fig. 1. Zn2⫹– cyclen–PAGE for heterozygosity screening.
(A and B), 107-bp PCR products (exon 10-2 in the online Data Supplement)
obtained with primers 5⬘-TGTCATCTTCCTGGGGTCCT-3⬘ and 5⬘-CCTTCTCCTCGGTCTCAGCG-3⬘ and containing the sequences of exon 10, as analyzed by
Zn2⫹– cyclen–PAGE with a discontinuous buffer system (A) and a continuous
buffer system (B). (C), 106-bp PCR products (exon 2-3 in the online Data
Supplement) obtained with primers 5⬘-AGTCCCTGGCAGCCATCGAG-3⬘ and 5⬘GGCCGGGGAGCCTCCTCCTC-3⬘ and containing the sequences of exon 2, as
analyzed with a discontinuous buffer system. (D), 100-bp PCR products (exon
21-3 in the online Data Supplement) obtained with primers 5⬘-CTGCTCAAGTGGGTGGCCTA-3⬘ and 5⬘-CCCTTCGGGTGCCCACACTC-3⬘ and containing the sequences of exon 21, as analyzed with a discontinuous buffer system. (E), 92-bp
PCR products (exon 20-2 in the online Data Supplement) obtained with primers
5⬘-TGGACACCACACAGGCCCCA-3⬘ and 5⬘-TGATGAATGTCTCGAACCAG-3⬘ and containing the sequences of exon 20, as analyzed with a discontinuous buffer
system. For all panels, lanes 1–10, Brugada patients; lane 11, IVF patient; lane
12, LQT3 patient; lanes 13–18, healthy controls.
phoresis with a discontinuous buffer system is apparent
in Fig. 1A compared with Fig. 1B [continuous buffer
system was 90 mmol/L Tris and 90 mmol/L borate (5 )].
Two additional differentially migrating bands representing heteroduplexes (lane 1 of Fig. 1A) were confirmed
more clearly by use of the newly modified Zn2⫹– cyclen–
PAGE. Subsequent direct sequencing of the DNA fragment including exon 10-2 of patient 1 revealed a heterozygous nucleotide substitution (G1212A) that does not affect
codon L404. A similar synonymous SNP (i.e., G87A in
codon A29) was also detected in exon 2 of 5 Brugada
syndrome patients and 2 healthy individuals (Fig. 1C).
We also detected a heteroduplex (lane 2 of Fig. 1D), which
we identified as a heterozygous G-to-A transversion in
the 5⬘ splice junction of the intron between exons 21 and
22, suggesting abnormal splicing linked to the Brugada
syndrome. No nucleotide change affecting the coding was
detected in the other Brugada patients. In some previous
studies, the reported incidence of SCN5A mutations in
Japanese patients with the Brugada syndrome varied
from 2% to 27% (14 ); therefore, further genetic testing will
be required to understand the genotype–phenotype relationship in this disease. Moreover, Zn2⫹– cyclen–PAGE
showed another abnormality in exon 20 of IVF and LQT3
patients (lanes 11 and 12 in Fig. 1E) attributable to a
common nucleotide alteration (G3575A) leading to a
codon mutation of R1192Q. This mutation has been reported to be associated with the Brugada (15 ) and LQT3
(16 ) syndromes. It is interesting to detect the same SNP in
an IVF patient without typical electrocardiographic signs
of the Brugada syndrome. No heterozygous mutation
associated with changes in the codon was detected in the
control individuals. Direct sequencing disclosed no mu-
2197
tation in the patient samples showing a single DNA band,
indicating that all heterozygous mutations in the SCN5A
gene tested were detected by our screening. Because the
samples showing a single DNA band in this study and a
previous report (5 ) had no mutations, the detection sensitivity of our screening would not be less than that
(generally 70%–90%) of other gel-based approaches such
as single-strand conformation polymorphism analysis
and denaturing gradient gel electrophoresis.
In conclusion, our Zn2⫹– cyclen–PAGE method is a
novel genetic approach to diagnosing autosomal dominant inherited diseases. Heteroduplexing with the homologous wild-type allele would also enable genotyping of
recessive-mode diseases. Use of the discontinuous buffer
system sharpened each DNA band in the minislab gel at
room temperature, making it easier to reliably detect
heteroduplex bands. Zn2⫹– cyclen–PAGE requires a general minislab PAGE system and an additive, Zn2⫹– cyclen,
without any special apparatus. The cost of Zn2⫹– cyclen
for 1 SCN5A gene (98 DNA fragments) was less than
$4.00 US dollars. This method may therefore be feasible
for initial screening of hereditary diseases in a clinical
laboratory.
We sincerely thank the individuals who provided blood
samples for this study. We also thank the Research Center
for Molecular Medicine, Graduate School of Biomedical
Sciences, Hiroshima University, for the use of their facilities. This work was supported by Grants-in-Aid for
Scientific Research (B) (15390013) and for Young Scientists
(B) (14770014) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.
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with MALDI-TOF mass spectrometry: practice, problems, and promise. Hum
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3. Hampe J, Wollstein A, Lu T, Frevel HJ, Will M, Manaster C, et al. An
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5. Kinoshita-Kikuta E, Kinoshita E, Koike T. A novel procedure for simple and
efficient genotyping of single nucleotide polymorphisms by using the Zn2⫹–
cyclen complex. Nucleic Acids Res 2002;30:e126.
6. Ornstein L. Disc electrophoresis. I. Background and theory. Ann N Y Acad Sci
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11. Kinoshita E, Kinoshita-Kikuta E, Koike T. A heteroduplex-preferential Tm
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12. Wang Q, Li Z, Shen J, Keating MT. Genomic organization of the human
SCN5A gene encoding the cardiac sodium channel. Genomics 1996;34:9 –
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13. Makielski JC, Ye B, Valdivia CR, Pagel MD, Pu J, Tester DJ, et al. A
ubiquitous splice variant and a common polymorphism affect heterologous
expression of recombinant human SCN5A heart sodium channels. Circ Res
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14. Hiraoka M. Inherited arrhythmic disorders in Japan. J Cardiovasc Electrophysiol 2003;14:431– 4.
15. Vatta M, Dumaine R, Varghese G, Richard TA, Shimizu W, Aihara N, et al.
Genetic and biophysical basis of sudden unexplained nocturnal death
syndrome (SUNDS), a disease allelic to Brugada syndrome. Hum Mol Genet
2002;11:337– 45.
16. Wang Q, Chen S, Chen Q, Wan X, Shen J, Hoeltge GA, et al. The common
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DOI: 10.1373/clinchem.2005.051011
Validation of the 99th Percentile Cutoff Independent
of Assay Imprecision (CV) for Cardiac Troponin Monitoring for Ruling Out Myocardial Infarction, Fred S.
Apple,1* Curtis A. Parvin,2 Kenneth F. Buechler,3 Robert H.
Christenson,4 Alan H.B. Wu,5 and Allan S. Jaffe6 (1 Hennepin
County Medical Center and University of Minnesota
School of Medicine, Minneapolis, MN; 2 Washington University School of Medicine, St. Louis, MO; 3 Biosite Incorporated, San Diego, CA; 4 University of Maryland School
of Medicine, Baltimore, MD; 5 San Francisco General
Hospital and the University of California School of Medicine, San Francisco, CA; 6 Mayo Clinic, Rochester, MN;
* address correspondence to this author at: Hennepin
County Medical Center, Clinical Laboratories P4, 701 Park
Ave., Minneapolis, MN 55415; fax 612-904-4229, e-mail
[email protected])
By definition, the probability that a single measured
cardiac troponin result exceeds the estimated 99th percentile is equal to 0.01 (1%) for a person randomly selected
from the general (reference) population. This is irrespective of the imprecision profile of the analytical method as
well as the lack of standardization of cardiac troponin
assays (1–7 ). However, the probability that a measured
result exceeds the 99th percentile limit for a specific
person from the reference population will vary depending
on that person’s true concentration and on the imprecision profile of the specific analytical method. Furthermore, the overall probability that at least one of multiple
measured values (the second or third measured cardiac
troponin concentration in a timed series of cardiac troponin orders) exceeds the 99th percentile also will vary
depending on the imprecision of the analytical method.
To investigate the influence of assay imprecision on
the likelihood of misclassifying healthy individuals or
patients without myocardial injury, we simulated the distribution of cardiac troponin I (cTnI) results in a general
population and added random analytical error reflecting
different assay imprecision profiles. One imprecision
profile assumes a CV of 37.5% at a cTnI of 0.05 ␮g/L,
decreasing to a CV of 25% at a cTnI of 0.07 ␮g/L, and a
CV of 9.4% at cTnI of 0.14 ␮g/L. The second imprecision
profile was obtained by multiplying the first imprecision
profile by a factor of 0.40, which produces a CV of 10%
at a cTnI of 0.07 ␮g/L. The distribution of “true” cTnI
concentrations in the general population was simulated
by generating 500 000 random values from an exponential
distribution. The mean of the exponential distribution
was selected so that the 99th percentile occurred at a cTnI
concentration of 0.07 ␮g/L for the case where the imprecision profile had a CV of 25% at 0.07 ␮g/L. For each of
the 500 000 true cTnI values, a gaussian-distributed random error was added that reflected the appropriate CV
given the imprecision profile and true cTnI concentration.
The distribution of values for a cTnI assay, assuming
an imprecision profile with a 25% CV at the 99th percentile limit of 0.07 ␮g/L, is shown in Fig. 1. The same
cardiac troponin distribution, assuming that the imprecision profile of this assay is improved, now with a 10%
CV at 0.07 ␮g/L, lowers the calculated 99th percentile
from 0.07 ␮g/L to 0.063 ␮g/L (Fig. 1, top). This demonstrates that when the imprecision of an assay is improved
at low concentrations, the 99th percentile limits will shift
to lower values because the width of the distribution is
reduced.
In a population of patients presenting with suspected
acute coronary syndrome who are being ruled out for
myocardial infarction, serial cardiac troponin orders at
presentation (0 h), 6 h, and 12 h are recommended (1, 2 ).
Also shown in Fig. 1 are the probabilities of at least 1 of 3
serially measured values exceeding the 99th percentile
limits as a function of the cTnI concentration for the 2
different imprecision profiles. The vertical lines are the
99th percentile limits for the 2 imprecision profiles. The
overall probability of a single measured result obtained at
a patient’s presentation (t ⫽ 0 h) exceeding the 99th
percentile limit is 0.01 for both imprecision values (Fig. 1,
middle panel). The probability of a cardiac troponin result
being falsely classified as positive (increased above the
99th percentile) during a second (t ⫽ 6 h) or third (t ⫽
12 h) measurement for the 2 different imprecision profiles
increases compared with the initial t ⫽ 0 h measurement.
The overall probability that a result exceeds the 99th
percentile is greater for the analytical method with a 25%
CV than for the analytical methods with the 10% CV as
follows: second measurement at t ⫽ 6 h, 0.016 vs 0.013;
third measurement at t ⫽ 12 h, 0.020 vs 0.015 (Fig. 1,
bottom panel). Thus, for 2 or 3 serial cardiac troponin
measurements in clinical practice, an additional 3 or 5 of
1000 patients, respectively, are likely to be misclassified as
false positives for the 25% CV imprecision assay. It should
be noted, however, that for the first measurement (t ⫽
0 h), 10 in 1000 patients will be misclassified as false
positive regardless of assay precision. We believe that the
clinical implications of this false-positive frequency are
insignificant.