Arch Irn Med 2001; 4 (4): 165-170

ORIGINAL ARTICLE
AMPLIFICATION REFRACTORY MUTATION SYSTEM
(ARMS) AND REVERSE HYBRIDIZATION IN THE
DETECTION OF BETA-THALASSEMIA MUTATIONS

Hossein Najmabadi PhD*, ** , Shahram Teimourian MS *, Talayeh Khatibi MD**,

Maryam Neishabury PhD**, Farzin Pourfarzad MS* **, Sayeh Jalil-Nejad MD*, Maryam Azad BS*,
Christian Oberkanins PhD***, Walter Krugluger MD PhD****

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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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Keywords Beta-thalassemia ARMS reverse hybridization

Introduction

eta-thalassemia is considered as the

most common autosomal single-gene
disorder worldwide. It can be found in
more than 60 countries with a carrier population of
up to 150 millions.1 At molecular level, betathalassemia represents a great heterogeneity as
more than 190 mutations have been identified for
the beta-globin gene responsible for this disease.2
The Mediterranean region, certain parts of North
Correspondence: H. Najmabadi MD, Karimi-Nejad Genetics and
Pathology Center, Shahrak-e-Gharb, 14667 Tehran, Iran. Fax: +9821-8077487, E-mail: Hnajm@Mavara.com.

and West Africa, Middle East, Indian subcontinent,

southern Far East and South East Asia have the
highest prevalence of the disease and comprise the
so-called thalassemia belt. The frequency and
spectrum of these mutations vary among different
populations. Immigration plays a major role in
both the distribution and the extent of mutation
variations within each country.3,4
Iran, with more than 25,000 affected
individuals, represents one of the areas in the
world with an unusually high prevalence of betathalassemia. The gene frequency of thalassemia
shows great variation within Iran from one area to
another. Provinces around the Persian Gulf and the

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Detection of Beta-Thalassemia Mutations

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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Caspian Sea with a gene frequency of more than

10% constitutes the thalassemia major zones in
Iran. Fars province in southern Iran shows a gene
frequency of 8 to 10 percent, whereas the
prevalence of the disease varies between 4% and
8% in other parts of the country.5-7
The use of sensitive and reliable diagnostic
methods plays a critical role in good screening and
therefore,
prevention
of
beta-thalassemia.
Regarding the heavy emotional and economical
burden imposed on the society, identification of
beta-thalassemia carriers and prenatal diagnosis of
the disease through an accurate and quick process
has been a major goal for researchers in Iran
during the past two decades.
During the last 8 to 10 years, amplification
refractory mutation system (ARMS) and restriction
fraction length polymorphism (RFLP) were the
principal techniques for diagnosis. However, Iran
with a multiethnic population of around 65 million,
represents a vast spectrum of beta-thalassemia

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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Table 1. Mutations investigated by ARMS and

reverse hybridization.

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1)Normal

M N

3)Major

M Marker

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Marker N

2)Minor

Internal control band

IVS 2.1 band

Figure 1. ARMS-PCR gel for beta-thalassemia diagnosis.

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H. Najmabadi, Sh. Teimourian, T. Khatibi, et al

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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A total of 8 to 10 specimens of EDTA blood

samples, amniotic fluid or chorionic villous
sampling (CVS) were obtained from randomly
selected clinically diagnosed beta-thalassemia
minor and major patients. DNA was extracted from
the samples using the saturated salt technique.7
In order to carry out ARMS, the DNA of
interest was first extracted. After DNA extraction,
PCR reactions were set up in two separate tubes
for each sample. One test tube for the amplification
of the normal ARMS primer and the second for the
amplification of the mutant ARMS primer. The
primers used for ARMS were kindly provided by
JM Old.9

Materials and Methods

PCR conditions for ARMS

A total of 20 L of final PCR reaction volume
was used for this purpose. The reaction volume
was composed of 0.5 micrograms of the DNA
template, 0.01 g of each of the four primers (2
control primers, 1 common primer, and 1 mutant/
normal ARMS primer for the normal/ mutant
reaction), 0.5 unit Taq DNA polymerase, and 0.2
mM of each dNTP in a solution of 10 mM tris-C1,
50 mM MgCl2, and 1 mM spermidine. The thermal
cycling regimen consisted of 30 cycles: preheating
at 94 C for 2 minutes, denaturing at 94 C for 1
minute, variable annealing, and extension at 72 C
for 1 minute.

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agarose gel. Presence of an amplified mutant the

ARMS primer product indicates presence of the
mutant allele. All samples contain an internal
control band. Sample 1 contains an amplified
product in the normal primer but lacks it in the
mutant primer; hence, implying a normal
individual. Sample 2 contains an amplified product
in both the normal and mutant primers; assigning
the individual of minor type. Sample 3 contains an
amplified product only in the mutant primer;
hence, it is the sample of an individual who is of
major type.

Figure 2. Reverse hybridization test strip.

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Detection of Beta-Thalassemia Mutations

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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18

remaining 2 samples showed IVS 2.1 mutations by

reverse hybridization but resulted in a falsenegative result with ARMS. ARMS was repeated
several times for these two samples. In one case,
after two rounds of repeating ARMS, we obtained
a positive result. In the other case, the DNA was
re-extracted to obtain a positive result. Of the ten
mutations and hemoglobin S that were screened in
this study, IVS2.1 was the most common mutation
detected, which is in accordance with a previous
study reported by Najmabadi, et al.7 A total of 78
mutant alleles have been detected. These included
29 IVS 2.1, 12 IVS 1.110, 7 codon 8, 7 codon 8/9,
6 IVS 1.6, 5 IVS 1.1, 3 codon 39, 3 IVS 2.745, and
1 codon 5 mutant allele (Table 2).

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single multiplex PCR reaction using three pairs of

primers and hybridized to pre-made test strips
containing the wild type and mutant-specific
probes for fourteen mutations, including HbS and
HbC. After several washes specifically bound
sequences were detected by enzymatic color
reaction (Figure 2).
Figure 2 shows examples of reverse
hybridization strips. The strip contains probes for
14 mutations. The right-hand box represents the
reference strip.10 On the left, the first case shows
no bands at any of the mutation sites, hence, she is
categorized as a normal individual. The second
case shows a single band at the codon 5 mutation
site. The third case shows two bands; one at codon
39 and one at HbS. The fourth case has a single
band at codon 8 while the fifth case shows a
double band, both at codon 8. This is the only
individual in this group which is of major type.
The sixth case displays a single band at codon 8 to
9 and the seventh case, a single band at intervening
sequence (IVS) 1.1. The eighth case represents a
single band at IVS 1.6, and the ninth case a band at
IVS 1.110. Finally, the tenth case has a single band
at IVS 2.1.

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

168 Archives of Iranian Medicine, Vol 4, No 4, October 2001

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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H. Najmabadi, Sh. Teimourian, T. Khatibi, et al

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

Depending on many factors

Very high

Number of PCR reactions per sample

8-88

25 or more (depending on the test strip)

Documentation

Requires documentation process after

experiment

Self-documented

1:1

10:1

Depending on the number of PCR

reactions

0.5 g genomic DNA for just one PCR

reaction

Technician time
(number of patients: time in days)
Starting material

Ethidium bromide (carcinogen)

Equipment

Expensive (large PCR machine, gel

electrophoresis, photodocumentation
system)

thalassemia test by reverse hybridization is only

one single PCR product of 5 L DNA. This is of
particular importance for prenatal diagnosis, where
there may not be enough DNA available to do 10
or more PCRs, which is a requierment performed
in ARMS.
Reverse hybridization is a more environmentfriendly technique as it avoids the use of
carcinogens such as ethidium bromide and
produces less toxic waste. From the economical
point of view, reduced labor time and using
simpler equipment decrease the cost of test per
sample by reverse hybridization.
In Iran, with its vast spectrum of betathalassemia mutations, a procedure such as reverse
hybridization is much more efficient and practical
than ARMS because as many as 25 betathalassemia mutations can be simultaneously
screened on a single strip in a reasonably short
time. This number can be increased by designing a
second strip to be run in parallel i.e. 50 mutations
on 2 strips.
Nevertheless, in other countries, such as
Cyprus, where only a few known mutations exist, a
procedure such as ARMS could be sufficient for
their needs. Table 3 compares the diagnostic
characteristics of the two techniques.
The existing version of the reverse
hybridization strip would cover 44% to 66%
(depending on the region of the country) of the
mutation spectrum in Iran. An extended version of
the reverse hybridization assay has recently been
released and covered 22 mutations, including

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single test strip by reverse hybridization, while in

the ARMS technique, 8 PCR reactions must be
performed for each sample to detect one single
mutation. These include two PCR reactions (using
mutant and normal primers) for the sample itself,
the negative control, the heterozygote positive
control and the homozygote positive control. It
means that with using ARMS and assuming every
PCR reaction works, we had to perform from 8 to
as many as 88 PCR reactions to screen for the 11
mutations in our study. Repeating a reverse
hybridization test under the same conditions
present no difficalty, whereas reproduction of
ARMS process is not as straight forward. Unlike
ARMS which requires a documentation process
after experimentation, reverse hybridization test
strips are self documented and thus, can be stored
and referred to with ease later, thereby reducing
possible record errors. These factors, as well as
increasing the accuracy of the tests, reduce our
turnover time from 2 to 3 days for ARMS to 6
hours for in reverse hybridization. This is of vital
importance in Iran, where the legislation allows
therapeutic abortion of an affected fetus only
within the first 16 weeks of gestation. In addition,
as most families consult a genetic diagnostic center
only after pregnancy, there is a limited window of
time available to detect the possible mutation
affecting the fetus and hence, a faster procedure
would provide families with sufficient time to
make appropriate decisions.
Furthermore, the amount of starting material
needed to perform a comprehensive beta-

None

Less expensive (small PCR machine,

agarose gel, small shaking water bath)

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Toxic materials

Number of mutations detected per test

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Detection of Beta-Thalassemia Mutations

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Lee GR, Forester J, Lukens J, Paraskovas F, Greer JP,

Rodgers GM. The Wintrobes Clinic Hematology. Vol 1.
10th ed. Baltimore: Lippincott, Williams and Wilkins;
1999.
Huisman THJ, Carver MFH. The beta- and deltathalassemia repository. Hemoglobin. 1998; 22: 169-95.
Lorey FW, Arnopp J, Cunningham GC. Distribution of
hemoglobinopathy variants by ethnicity in multiethnic

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Vetter B, Schwarz C, Kohne E, Kulozik AE. Betathalassemia in the immigrant and non-immigrant German
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5 Habibzadeh F, Yadollahie M, Merat A, Haghshenas M.
Thalassemia in Iran: an overview. Arch Irn Med. 1998; 1:
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6 Mahboudi F, Zeinali S, Merat A, et al. The molecular
basis of -thalassemia mutations in Fars province, Iran.
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Hemoglobin. 2000. [Accepted for publication].
8 Newton CR, Graham A, Hepatinstall LE, et al. Analysis
of any point mutation in DNA. The amplification
refractory mutation system (ARMS). Nucleic Acid Res.
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9 Old JM, Varawalla NY, Weatherall DJ. Rapid detection
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10 Vienna Laboratories. -Globin Strip Assay. 1998.

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codon 22 (7 bp del), IVS 1.25 (25 bp del), codon

30 (G->C), codon 36/37 (-T), codon 44 (-C), and
IVS 1.116 (T->G), mutations that are frequently
found in the Iranian population.
In conclusion, with the addition of probes for
these new mutations to the reverse hybridization
test strip, we believe that this technique is the best
option that can be employed for carrier detection
and prenatal diagnosis of beta- thalassemia in Iran.

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