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ORIGINAL ARTICLE
AMPLIFICATION REFRACTORY MUTATION SYSTEM
(ARMS) AND REVERSE HYBRIDIZATION IN THE
DETECTION OF BETA-THALASSEMIA MUTATIONS
SI
Karimi-Nejad Genetic and Pathology Center, ** University of Welfare and Rehabilitation Sciences,
Tehran, Iran, *** Vienna Lab Labordiagnostika GmbH,
**** Institute of Clinical Chemistry, Rudolfstiftung Hospital, Vienna, Austria
Abstract
Background-Beta-thalassemia is the most common hereditary disorder in Iran and
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during the past 10 years, amplification refractory mutation system (ARMS) and restriction
fragment length polymorphism (RFLP) were the sole molecular technique used for diagnosis
of the disease. Although many beta-globin gene mutations exist in the Iranian multiethnic
population, these techniques seem labor-intensive, time-consuming and expensive. This
has urged us to use new techniques such as reverse hybridization and direct sequencing
this issue.
Methods-In this study, reverse hybridization was applied in parallel with ARMS to
screen for the 10 most common beta-thalassemia mutations and hemoglobin S in 82
patients clinically diagnosed as beta-thalassemia minor and major.
Results-From the 82 cases detectable by both methods, 80 had similar results.
Compared to ARMS, reverse hybridization appeared to be more reliable, cost-effective, fast
and applicable.
Conclusion-Considering the vast spectrum of beta-thalassemia mutations in Iran, a fast
and reliable technique such as reverse hybridization represents vital advantages in
comparison with the traditional diagnostic methods. In fact, it is recommended as the
technique of choice that can be employed by the National Thalassemia Project for the
detection and prenatal diagnosis of beta-thalassemia in Iran.
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IVS 1.1 (G to A)
Codon 8 (-AA)
IVS 1.110 (G to A)
Codon 39 (C to T)
IVS 1.6 (T to C)
IVS 2.745 (C to G)
IVS 1.5 (G to C)
Codon 5 (-CT)
ARMS
ARMS is a PCR-based method, which uses
allele-specific priming. In this method, an oligonucleotide primer with a triple end complementary
to the sequence of a specific mutation, coupled
with a common primer is used in one PCR
reaction. In parallel, a corresponding normal
primer coupled with a common primer is used in
another PCR reaction. The presence of an
amplified product in the first reaction indicates the
presence of the mutation while its absence suggests
presence of the normal DNA sequence at that
specific site. In the second reaction the presence of
an amplified product suggests presence of a normal
DNA sequence at that specific site while its
absence suggests presence of the mutation8 (Figure
1).
Figure 1 shows ARMS-PCR turn on a 2%
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IVS 2.1 (G to A)
Codon 8/9 (+G)
mutations, making these techniques too laborintensive, slow and expensive. Therefore, in recent
years modern molecular biology techniques, such
as reverse hybridization and direct sequencing,
have been implemented for the diagnosis of this
disease. We report the application of reverse
hybridization for detection of hemoglobin S and
the 10 most common mutations (Table 1) and
demonstrate its advantages versus ARMS in the
detection and prenatal diagnosis of betathalassemia mutations as well.
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1)Normal
M N
3)Major
M Marker
Ar
Marker N
2)Minor
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Electrophoresis conditions
Fifteen microliters of the PCR products were
removed and mixed with 3 L of a loading buffer
and then loaded on a 2% agarose gel. The gel was
set at 100 volts for 1 hour and then stained with
ethidium bromide. After staining, the bands could
be seen under UV light.
Reverse hybridization
For reverse hybridization, a commercially
available assay (-globin) strip assay (Vienna Lab,
Austria)10 was used according to the instructions
provided by the manufacture. The -globin regions
of interest were amplified from isolated DNA in a
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Table 2. Number of specific mutations detected by ARMS and reverse hybridization (RH) in DNA samples
extracted from blood, chorionic villi (CV) and amniotic fluid (AF) of beta-thalassemia patients.
Blood
CV
AF
ARMS
RH
ARMS
RH
ARMS
RH
IVS 2.1
29
17
19
Codon 8/9
IVS 1.110
12
IVS 1.6
IVS 1.5
IVS 1.1
Codon 8
Codon 39
IVS 2.745
Codon 5
Total
78
52
54
18
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Total number of
mutant alleles detected
Mutation
Results
Of the 82 samples detectable by both ARMS
and reverse hybridization, 80 gave identical results
in both methods. Specifically, the mutations
detected in both were identical. However, the
Discussion
Currently, over 25,000 individuals with betathalassemia exist in Iran.5 Prenatal diagnosis is the
key to prevention and control of this disease. In
this study we introduced the application of reverse
hybridization for the detection of beta-thalassemia
mutations and compared it with ARMS, which is
one of the most commonly used techniques for
diagnosis of this disease in Iran. The most common
mutation detected was IVS2.1. This is in
accordance with our previous findings.7
Our results suggest that reverse hybridization is
a more reliable technique that can also reduce
false-negative results. Only one PCR reaction is
required to screen for more than 20 mutations on a
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Table 3. Comparison of different factors determining the efficiency of ARMS and reverse hybridization in
beta thalassemia diagnosis.
Turnover time
ARMS
Reverse hybridization
several days
6-8 hours
Specificity
High
High
Reaction reproducibility
Very high
8-88
Documentation
Self-documented
1:1
10:1
Technician time
(number of patients: time in days)
Starting material
Equipment
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None
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References
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