AMERICAN JOURNAL BIOTECHNOLOGY AND MOLECULAR SCIENCES

ISSN Print: 2159-3698, ISSN Online: 2159-3701, doi:10.5251/ajbms.2013.3.1.24.28

© 2013, ScienceHuβ, http://www.scihub.org/AJBMS

Molecular diagnosis of hemoglobinopathies using allele-specific

polymerase chain reaction in Nigeria
1*
Akanni E.O, 2Alli O.A.T and 3Mabayoje V.O
1
Hematology Division, Biomedical Science Department, College of Health Sciences,
Ladoke Akintola University of Technology, P.M.B 4400, Osogbo. Nigeria.
2
Biomedical Science Department, College of Health Sciences, Ladoke Akintola
University of Technology, P.M.B 4400, Osogbo. Nigeria.
3
Department of Hematology and Blood transfusion, Ladoke Akintola University of
Technology, P.M.B 4400, Osogbo. Nigeria.
*Correspondences: Dr. Akanni E. Olufemi. Department of Biomedical Science,
College of Health Sciences, Ladoke Akintola University of
Technology, P.M.B 4400, (OS 230001).Osogbo. Nigeria
Telephone: +234-803-360-0747., E- mail: eoakanni@lautech.edu.ng or
olufemiakanni@yahoo.com
ABSTRACT
Hemoglobinopathies especially sickle cell anaemia is a serious health problem in Nigeria. The
need for an accurate and reliable method for early detection cannot be over emphasized for an
effective control. A study on the molecular identification of hemoglobin types A, S and C in
neonates and adult subjects using allele-specific Polymerase Chain Reaction (AS-PCR) was
carried out. Blood samples from 200 neonates and adults who served as controls were run blindly
for both AS-PCR and conventional cellulose acetate paper electrophoresis. Each extracted DNA
sample was subjected to AS-PCR using two primer sets HbA and HbS separately and HbA and
HbC primer sets owing to varying PCR conditions. Visible bands were seen at 203 bp regions for
both HbA and HbS alleles and at 216 bp regions for HbC alleles and the hemoglobin phenotypes
obtained. Results also revealed that hemoglobins A, S and C identified from adult blood samples
using AS-PCR correlated well with those obtained using conventional hemoglobin electrophoresis
(t test; p > 0.05) while there was discrepancy in the results obtained using neonates samples with
AS-PCR predicting the genotypes correctly. The study thus demonstrates the accuracy and
precision of allele-specific PCR for the diagnosis of hemomoglobinopathies.
Keywords: Alleles, polymerase chain reaction, hemoglobinopathy, sickle cell anaemia,
hemoglobin electrophoresis, DNA.
INTRODUCTION: Cellulose acetate electrophoresis is very useful for
quick screening of a small number of samples but the
Hemoglobinopathies especially sickle cell anaemia is
protein bands are relatively wide and abnormal
a serious health problem in Nigeria. World Health
hemoglobins overlap. Also, quantitative densitometry
Organization (WHO) report showed that an average
of abnormal hemoglobins is inaccurate at low
of 150,000 infants is born with sickle cell disease in
concentrations (i.e HbA2, HbF). Since a great
Nigeria. The country also ranks first as the sickle cell
percentage of hemoglobin in newborns is HbF,
endemic country in the world with an annual infant
cellulose acetate electrophoresis is not a reliable
death of 100,000, representing 8 per cent of infant
technique for the determination of genotype in
mortality in the country [1].
neonates [3].
In the United States, hemoglobin SS disease (sickle
Despite the improved sensitivity of DNA-based
cell anemia) affects mostly African Americans.
testing methods, quantities of obtainable foetal DNA
However, forms of sickle cell disease may occur in
were often insufficient. Sensitivity was greatly
people with different ethnic backgrounds, such as
enhanced by the development of polymerase chain
those whose ancestors came from Mediterranean
reaction (PCR), a method of in-vitro DNA
countries, East India, or Middle Eastern countries [2].
amplification that employs repeated cycles of

denaturation, annealing of oligonucleotide primers to
the target DNA, and enzymatic primer extension to
amplify DNA flanked by the primers [4].
Allele-specific polymerase chain reaction (AS-PCR)
is a convenient method for genotyping Single
Nucleotide Polymorphisms (SNPs) and mutations.
AS-PCR is also known as amplification refractory
mutation system (ARMS). This technique is a quick
and dependable genotyping protocol that requires
minimal instruments found in most laboratories. It is
based on the extension of primer only when its 3'-end
is a perfect complement to the allele present in the
input sample. Thus, if a single base polymorphism
occurs, the genotyping results can be observed by
simply comparing the length of PCR products [5].
The rapid, non-radioactive approach to the diagnosis
of sickle cell anemia allows the direct detection of the
normal or the sickle cell β-globin allele in genomic
DNA without the additional steps of probe
hybridization, ligation or restriction enzyme cleavage
[6]. Several laboratories in the developed countries
have been using this technique [7-9] in diagnosing
hemoglobinopathies in neonates in absence of full
foetal hemoglobin conversion to adult form. Hence
the need to apply the technique in Nigerian
environment where the prevalence is high and there
is need for pre-natal diagnosis. A study on the
molecular identification of hemoglobin types A, S and
C in neonate and adult subjects using allele-specific
Polymerase Chain Reaction (AS-PCR) was carried
out.
In this study, the allele-specific polymerase chain
reaction was employed for the detection of the single
th
nucleotide polymorphism and mutation in the 6
position of the β globin gene that causes
hemoglobins S and C disease (as a result of valine
and lysine substitution, respectively) in neonates. The
results will be compared with those obtained using
the convectional cellulose acetate paper hemoglobin
electrophoresis in adult samples as neonatal samples
are not reliable for cellulose acetate method. Part of
this study was presented as poster and awarded a
Berend Houwen Travel grant at the XXIVth
International Symposium on Technological
Innovations in Laboratory Hematology (by the
International Society for Laboratory Hematology-
ISLH) in New Orleans, United States of America.
MATERIALS AND METHODS:
Study sites: One hundred blood samples were
selected from neonates samples aged between 1 and
15 days old from routine blood samples sent from
25
Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 24-28
Paediatrics clinics of Ladoke Akintola University of
Technology Teaching Hospital, Osogbo, Osun State,
Nigeria and Osun State General Hospital, Asubiaro,
Osogbo, Osun State, Nigeria to the laboratory
facilities for routine investigations. One hundred
blood samples were also selected from adults blood
samples sent for routine investigations. The samples
were analyzed at the Molecular Biology Laboratory of
the college of health sciences, Ladoke Akintola
University of Technology, Mercyland Wing, Osogbo,
Osun State, Nigeria.
DNA EXTRACTION
DNA was extracted from buffy coat prepared from
blood collected from all subjects using Ultraclean
Bloodspin DNA Extraction Kit (Mo Bio Laboratories
Inc., USA) following manufacturer’s instructions.
PCR AMPLIFICATION
Each DNA sample was subjected to allele-specific
PCR using two primer sets HbA and HbS separately
and HbA and HbC primer sets owing to varying PCR
conditions. Hβ14A (5’-CACCTGACTCCTGA-3’) and
BGP2 (5’-AATAGACCAATAGGCAGAG-3’) were
used as the primer set for the amplification of the
normal β-globin gene (HbA primer set). Similarly,
Hβ14S (5’-CACCTGACTCCTGT-3’) and BGP2 (5’-
AATAGACCAATAGGCAGAG-3’) were used as the
primer set for the amplification of the sickle cell gene
(HbS primer set). Reactions were carried out for 35
cycles at optimal PCR conditions. Hemoglobin C
primer set, beta 5’-a (5’-
GTACGGCTGTCATCACTTAGACCTCA-3’) and beta
A–b (5’-TAACGGCAGACTTCTCCTC-3’),
corresponding to the wild-type allele, and in the other,
the primer used were beta 5’- a and beta C-b (5'-
TAACGGCAGACTTCTCCTT-3'), corresponding to
the hemoglobin C allele. All the mix was run on the
GeneAmp PCR System 9700 machine (Applied
Biosystems, UK). Agarose gel electrophoresis was
run on the PCR products with DNA bands visualized
and photographed by filtered ultraviolet (UV)
illumination on the Syngene gel documentation
system (Syngene, UK).
Electrophoresis was carried out in 0.7% agarose slab
gels in tris-borate buffer (89 mM Tris, 89 mM boric
acid and EDTA pH 8.0) containing about 0.5 µg/ml of
ethidium bromide to enhance visualization of DNA
bands under UV light. The respective PCR products
were mixed with 10 µl of loading buffer, loaded and
run on the electrophoresis machine at 70 V for 1
hour. The 100 bp DNA ladder was mixed with 10 µl of
loading buffer and also loaded on a slot on the
 Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 24-28

agarose gel. Sigma Gel Loading Solution Type I was
used as the loading buffer. It contains 0.25 % (w/v)
Bromophenol blue, 0.25 % (w/v) Xylene cyanole FF
and 40 % (w/v) sucrose in water.
The size of DNA product (203 bp for Hb A and S;
216bp for Hb C) were read against the 100 bp DNA
ladder and genotype results were obtained.
Cellulose acetate paper electrophoresis: Lysate of
each sample was made by washing the sample four
times in saline, and haemolysed in carbon
tetrachloride. The haemolysate of each sample was
loaded on the cellulose acetate paper along with
control samples such as Hb AS and AC. The 250-350
V was applied for 20 minutes or until visible and clear
separation was obtained [10].
Statistical Analysis: Paired ‘t’ test was used for the
analysis of difference between the results. P < 0.01
indicates significance
RESULTS AND DISCUSSION:
Visible bands were seen at 203 bp regions for both
HbA and HbS alleles and at 216 bp regions for HbC
alleles and the hemoglobin phenotypes obtained as
shown in Figure 1.

203 bp
500 bp
100
bp
1A 1S 2A 2S 3A 3S 4A 4S 5A 5S 6A 6S 7A 7S SM

Fig1. Molecular identification of hemoglobins A and S using Allele specific PCR.

The hemoglobin phenotype of the above 7 individuals are: Samples 1,3,4,6 and 7 – HbAA; Sample 2 – HbAS and sample 5
– HbSS.

500 bp

216 bp
100 bp

1A 2A 3A 4A 5A NC SM 1C 2C 3C 4C 5 NC SM

Fig 2. Molecular identification of Hb A and C using AS-PCR.

Lanes 1,2,3,4,5 are for HbA on the left side of size marker while Lanes 1,2,3,4 on the right side of SM are identified as
th
HbC. There is no amplification on the 5 lane. NC; negative control
SM –size marker.

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Table 1: Comparison of AS - PCR and blindly run conventional cellulose acetate paper hemoglobin electrophoresis
on adult control samples
Hb genotype AS-PCR
electrophoresis
AC 09
AA 53
SC 05
SS 08
AS 25
Values are number of Hb genotypes per method; n = 100.
Results also revealed that hemoglobins A, S and C
identified from adult blood samples using allele-
specific PCR correlated well with those obtained
using conventional hemoglobin electrophoresis.
There was no statistical difference (t- test; p > 0.01)
between the two methods (Table 1). The study thus
demonstrates the accuracy and precision of allele-
specific PCR for the diagnosis of sickle cell anaemia
and other forms of hemoglobinopathies in the study
population. The method is convenient and also safe
since the result is obtained without the use of
radioactivity. AS-PCR technique could therefore be
conveniently applicable to prenatal diagnosis of
hemoglobinopathy and screening of neonates since
patients’ or clients’ DNA is required for analysis
unlike the conventional cellulose acetate paper
electrophoresis method that requires erythrocytes’
hemoglobin.
Following successful determination of accuracy and
precision of the AS-PCR method in identification of
hemoglobins A, S and C in adults, the method was
reliably applied to screen the 100 neonate samples
classifying them into; 21% AS, 7% AC, 62% AA, 6%
SS and 4% SC. However, using the convectional
cellulose acetate paper electrophoresis method to
identify the various hemoglobin types in neonate
samples produced inconsistent results contrary to the
AS-PCR owing to the incomplete fetal-adult
hemoglobin conversion. Hemoglobin electrophoresis
presents a 60% and 65% correlation with AS-PCR
results in typing Hb AA and Hb AC respectively. AS-
PCR therefore remains the golden standard for
determining Hb genotype in neonates and for
prenatal diagnosis.
In view of the high prevalence of sickle cell anaemia
in Nigeria, AS-PCR technique which is still relatively
Am. J. Biotechnol. Mol. Sci., 2013, 3(1): 24-28
Acetate paper Students t-test P- value
9 >0.01
53 >0.01
5 >0.01
8 >0.01
25 >0.01
new in the country could be applicable to pre-natal
diagnosis of sickle cell anaemia to assist in early
detection and onset of interventory measures.
However, the prospect of abortion as a solution to
homozygous ‘S’ detection, remains a strong issue of
religious and ethical concern in Nigeria.
ACKNOWLEDGEMENT: The molecular biology
works were all carried out in the Molecular Biology
Laboratory, Department of Biomedical Sciences,
Ladoke Akintola University of Technology, Mercyland
Campus, Osogbo. We would like to thank the Ex-Vice
Chancellor (Professor B.B.A. Adeleke) and
Management of Ladoke Akintola University of
Technology for the establishment of the Molecular
Biology Laboratory. The authors are grateful to the
International Society for Laboratory Hematology
(ISLH) for the Berend Houwen travel award granted
this work at the ISLH 2011 conference in New
Orleans, USA. We also acknowledge Mr Oyenike for
his technical assistance.
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