Yonago Acta medica 2009;52:121–125

Sensitivity and Specificity of Denaturing HPLC to Detect
MYBPC3 Gene Mutations in Hypertrophic Cardiomyopathy
Udin Bahrudin*†, Einosuke Mizuta*, Mahayu Dewi Ariani‡, Yora Nindita* and Ichiro
Hisatome**Division of Regenerative Medicine and Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Yonago 683-8503, †Center ofBiomedical Research, Faculty of Medicine, Diponegoro University, Semarang 50231, Indonesia
and ‡Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori
University, Yonago 683-8503, Japan

Denaturing high-performance liquid chromatography (DHPLC), which has recently beendeveloped as an automated method to detect mutations, is at least ten times less expensive
than the direct sequencing method; however, its sensitivity and specificity for cardiac myosin-binding protein C (MYBPC3) gene mutations in hypertrophic cardiomyopathy have notbeen reported yet. A mutation analysis of exons 1 to 35 of MYBPC3 gene from 20 Japanese patients with hypertrophic cardiomyopathy was performed using DHPLC and direct
sequencing. Compared to direct sequencing, the sensitivity and specificity of DHPLC
were 87.5 % and 97.42 %, respectively. Its positive and negative predictive values were
41.18 % and 99.74 %. The positive and negative likelihood ratios were 33.95 and 0.13, andthe prevalence was 2.02 %. DHPLC showed high sensitivity and specificity for detecting
MYBPC3 gene mutations in hypertrophic cardiomyopathy. The use of this complementary
sequencing method should reduce the cost of detection of MYBPC3 gene mutations, andcould be used to screen patients with hypertrophic cardiomyopathy.
Key words: cardiac myosin-binding protein C gene; denaturing high-performance liquid chromatography; hypertrophic cardiomyopathy; mutation detection

Genotyping of sarcomeric genes is currently used
to identify mutations in patients with familial hypertrophic cardiomyopathy; hence, it is essential
to have an automatic, inexpensive, non-hazardous,
highly sensitive and specific method for genetic
analysis. Hypertrophic cardiomyopathy is inherited as a Mendelian-autosomal dominant trait andis caused by mutations in any of the 13 genes encoding proteins of the cardiac sarcomere and thenon-cardiac sarcomeric protein (Seidman et al.,
2001; Richard et al., 2006). In several studies, mutations of the cardiac myosin-binding protein C gene

(MYBPC3) were found to be one of the two mostcommon genetic causes of familial hypertrophic cardiomyopathy (Jääskeläinen et al., 2002; Erdmann etal., 2003; Mörner et al., 2003; Richard et al., 2003).
To date, more than 150 mutations, consisting of missense, deletion/insertion and splice site abnormalities, have been identified in MYBPC3.
Direct sequencing is the gold standard method
for the detection of gene mutations, but it is laborious, time consuming and expensive (Suzuki et al.,2009). Alternative mutation screening techniques
may play a role in both candidate gene analysis andAbbreviations: DHPLC, denaturing high-performance liquid chromatography; MYBPC3, cardiac myosin-binding protein C gene121

U. Bahrudin et al.

diagnosis. Ideally, a method to screen mutation in
new samples should be sensitive, non-hazardous,
relatively inexpensive and fully or at least semiautomatic to minimize the time required for the
test, labor and cost. Denaturing high-performanceliquid chromatography (DHPLC) has recently
been developed as an automated method to detectmutations, and it is at least 10 times less expensive
than the direct sequencing method (O’Donovan etal., 1998). Therefore, it potentially overcomes the
drawbacks of the direct sequencing method. However, the sensitivity and specificity of DHPLC todetect MYBPC3 gene mutations in hypertrophic
cardiomyopathy have not been reported yet. In
this study, we analyze MYBPC3 gene in patientswith hypertrophic cardiomyopathy and asses thesensitivity and specificity of DHPLC by comparing
them with those of the direct sequencing method.

melting temperatures were chosen from the predictions. Three types of samples were chosen for
DHPLC, i.e., wild type as a control, samples fromhypertrophic cardiomyopathy patients, and a mixof wild type and patient samples. The heteroduplex
peak resolve was influenced by the specific nucleotide mismatch present and the melting characteristics of the surrounding bases. Elution profiles
that differed from wild type indicated the presenceof mutation or polymorphism. Normal samples
were recruited for control (wild type) amplicon of
DHPLC analysis from 5 healthy Japanese. The
sequencing was performed with the Abi Prism 373
DNA Sequencer (Applied Biosystems, Foster City,CA) according the manufacturer’s instructions.
This study has been approved by the Ethical Committee of Faculty of Medicine, Tottori University.
The diagnostic test was based on a 2X2 contingency table analysis to compare DHPLC and thesequencing method. Since the sample ampliconof DHPLC and sequencing was a DNA fragmentfrom each exon of the MYBPC3 gene, the sample
unit which was used for statistical analysis waseach exon of MYBPC3. The estimated number ofmutations of the MYBPC3 gene in hypertrophic
cardiomyopathy is 1 mutation in each patient.
Thirty three fragments of PCR product were obtained from 35 exons of MYBPC3 (Fig. 1A). Exon
2 was amplified in 2 separated fragments. Three
separated fragments were obtained from 3 of 2
short tandem exons. They were exon 14–15, exon
19–20 and exon 34–35. Using the formula of single
proportion, 12 positive signals of DNA amplicon
were required in DHPLC, and the minimum total
number of DNA amplicons was 369.

Subjects and Methods
The subjects were 20 Japanese patients diagnosed
as having primary hypertrophic cardiomyopathy
at Tottori University Hospital. Briefly, clinical diagnostic criteria were adults with maximum myocardium wall thickness of > 13 mm determined byechocardiography in the absence of any other confounding diagnosis. Genomic DNA was extracted
from peripheral white blood cells using the standard phenol-chloroform procedure. Exons 1 to 35of the MYBPC3 gene were amplified by PCR using
primers designed according to the published genomic sequences of MYBPC3 (Carrier et al., 1997).
PCR products were run in 1% agarose gel, stainedwith ethidium bromide and visualized employing a
UV transilluminator.
DHPLC analysis was performed using theTransgenomic WAVE system (Transgenomic,
Omaha, NE) to detect genetic variants. To obtainheteroduplexes with the WAVE system, the prediction of temperature and gradient condition was determined using the Stanford Genome Technology
Center software (http://insertion.stanford.edu/melt.
html) and the WAVEMAKER software. Three

Results and Discussion
All MYBPC3 exons were successfully amplified
by PCR at 60˚C melting temperature (Fig. 1A).
DHPLC was performed for MYBPC3 gene DNA
from all 20 patients and 5 normal subjects. Waves
with 2 or more peaks in the amplicon from patient samples indicated a heterozygous mutation or122M 1 2a 2b 3

4

5

6

7

8 9 10 11 12 1316

M 17 18

21 22 23 24 25 26 27 28 29 30 31 32 33

34 – 35bp

19 –20A

14 –15

Mutation detection in HCM by DHPLC

1357—
603—
310—

B

Wild type

Sample from patient

Mix
Heterozygous

Homozygous

Fig. 1.
A: PCR amplification product of MYBPC3 gene from exon 1 to exon 35. Exon 2 was amplified using 2 different primer
sets; each of exons 14 and 15, 19 and 20, and 35 and 36 were amplified using one primer set. All MYBPC3 exons weresuccessfully amplified by PCR at 60˚C melting temperature.
B: An example of DHPLC wave analysis from the same segment of amplified DNA. Double-peak waves in the ampliconfrom a patient DNA sample indicated a heterozygous mutation or polymorphism, whereas, a wave with two or more
peaks in the mix amplicon indicated a homozygous mutation or polymorphism.
DHPLC, denaturing high-performance liquid chromatography; M, molecular weight marker.

Table 1. Sensitivity and specificity of DHPLC
versus sequencing

polymorphism, whereas, in the mix amplicon they
indicated a homozygous mutation or polymorphism(Fig. 1B). We found more than one peak of the
DHPLC wave at exon 5 of samples from both control subjects and patients indicating the probabilityof polymorphism.
Sequencing was performed for all DNA
samples from the patients. Consistent with the
DHPLC result, we found a polymorphism in exon
5 of MYBPC3 gene in samples from patients andnormal subjects, and therefore, we excluded exon
5 from the analysis of sensitivity and specificity of
DHPLC versus sequencing. Three hundred sixty
nine DHPLC waves out of 660 were reliable for the
analysis of sensitivity and specificity of DHPLC
versus sequencing. As shown in Table 1, one false

DHPLC (+)
DHPLC (–)
Total

Sequencing(+)

Sequencing
(–)

Total

7
1
8

10
378
388

17
379
396

DHPLC, denaturing high-performance liquid chromatography.
Sensitivity = 87.5%, specificity = 97.42%, positive predictive value = 41.18%, negative predictive value = 99.74%,
positive likelihood ratio = 33.95, negative likelihood ratio= 0.13, prevalence = 2.02%.

negative and 10 false positive were identified by
DHPLC. The sensitivity and specificity of DHPLC were 87.5 % and 97.42 %, respectively. In
123

U. Bahrudin et al.

addition, its positive and negative predictive values
were 41.18 % and 99.74 %, the positive and negative likelihood ratios were 33.95 and 0.13, and theprevalence was 2.02 %.
Since a practical application of DHPLC genetic analysis would be screening to reduce unnecessary sequencing, it is important that its sensitivity
be the highest obtainable. In several studies todetect mutations in various genes, the sensitivityof DHPLC consistently exceeded 96 % (Underhill
et al., 1997; Liu et al., 1997-1998; Wagner et al.,1999a, 1999b; Xiao et al., 2001). The ability of
DHPLC to detect a mutation depends upon theformation of heteroduplexes (Xiao et al., 2001;
Rudolph et al., 2002); the mixed amplicon shouldbe tested using various ratios. For optimal mutation detection, it was important to evaluate the PCR
reaction and running profile. In this study, we used
Taq DNA polymerase for the PCR reaction andsequencing. The sensitivity and specificity of DHPLC may be increased by using other polymerases
which have higher fidelity. This warrants further
study.
Identification of mutations in sarcomericgenes is meaningful for patients with hypertrophic
cardiomyopathy. Once a mutation is identified in apatient, screening of all family members is recommended, particularly for those who have symptoms
of cardiac disease. For this reason, it is importantto establish an inexpensive method that improves
screening of MYBPC3 gene mutations in hypertrophic cardiomyopathy.
Mutation analysis using sequencing is expensive because each exon is sequenced in both
directions. The cost per run, even in subsidized
laboratories, is about US $10 to $25, excluding thecosts of DNA isolation, PCR reagents and labor/time(O’Donovan et al., 1998; Xiao et al., 2001; Suzuki etal., 2004). Any mutation screening must be technically simple, use inexpensive reagents, and be highly
specific and highly sensitive. Therefore, a mutation screening method may contribute to reduce thenumber of unnecessary sequencing tests. DHPLC isa relatively new method to detect mutations. It is run
automatically and it is relatively inexpensive. Eachamplicon of DHPLC is screened for about US $1.5
(Wagner et al., 1999). This inexpensive method canbecome affordable worldwide, particularly for early
genetic diagnosis or gene mutation screening.
Finally, we conclude that because of the highsensitivity and specificity of DHPLC regarding detection of MYBPC3 gene mutations in hypertrophic
cardiomyopathy, it should become a useful mutation screening method for hypertrophic cardiomyopathy.
Acknowledgments: We thank to Prof. Eiji Nanba and Ms.
Kaori Adachi for technical assistance. This study was
supported by a grant-in-aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan (18590775) (awarded toProf. Ichiro Hisatome). Dr. Udin Bahrudin was a fellow
researcher supported by the Venture Business Laboratory,
National University Corporation Tottori University.
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Received October 15, 2009; accepted November 6, 2009
Corresponding author: Udin Bahrudin, MD, PhD

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