Evaluation of a Single Tube Multiplex PCR for rapid detection of common α+

thalassemia deletions in Indian population

α thalassemia is highly prevalent and much overlooked disorder in the Indian

subcontinent. Diagnosis of α thalassemia rests on the detection of genetic mutations. Till
date, Southern Blot hybridization and Gap-PCR have been used in Indian setup to
delineate the two most common α thalassemia mutations prevalent, i.e. –α3.7 and – α4.2.
Southern Blot hybridization is a time-consuming and complex process requiring use of
radioactive probes and therefore not practical for most of the laboratories. Gap-PCR
considerably simplified the detection however it requires multiple tube amplifications for
precise characterization of the two major –α3.7 and – α4.2 alleles which is cumbersome for
large scale screening.
Thus in an effort to develop a technique suitable for population screening, a multiplex
PCR amplification procedure carried out in a single tube has been developed.
This study was undertaken to evaluate the efficacy of Single Tube Multiplex PCR
technique to detect the α+ thalassemia deletions in Indian population. 86 subjects of β
thalassemia trait were screened for
–α3.7 and – α4.2 deletions using this technique. 12 out of 86 (14%) cases were positive for –
α3.7, out of which 3 were homozygous for this deletion (–α3.7/ –α3.7). all the samples were
negative for – α4.2 deletion. We found Single Tube Multiplex PCR a simple, rapid and
efficient method for detection of the common α+ thalassemia mutations in India. It should
considerably facilitate large scale population screening and prenatal diagnosis of α+
thalassemia.

Introduction

The α thalassemia are the most common single gene disorders in the world, and are
characterized by the reduction or complete absence of α gene expression.
α thalassemia most frequently result from the deletion of one, α+ (-α) or both, α0 (--) α
genes from the normal complement of two α globin genes (α α) present on the short arm
of chromosome 16(α α/ α α). α thalassemia occurs at a high frequency with prevalence
ranging from 10 – 97% in several ethnic groups and tribal populations of India. However
despite the high prevalence of α thalassemia in India, large scale population screening
programmes have not yet been established. Diagnosis of α thalassemia poses a great
challenge as most cases of heterozygous α thalassemia only result in varying degrees of
hypochromic anemia which are very frequently mistaken for Iron deficiency anemia.
DNA analysis remains the only way to validate its diagnosis as no definitive
hematological test is available. PCR based methods have made the detection relatively
simple and accurate. However it requires multiple tube amplifications for precise
characterization of mutations, which is rather cumbersome for large epidemiological
screening. Recently a single tube multiplex PCR amplification procedure has been
evolved, using a single tube for simultaneous detection of both the common deletional
forms of α+ thalassemia –α3.7 and – α4.2. The aim of this study was to evaluate the
efficacy and practicality of single tube multiplex PCR for the simultaneous detection of
α+ thalassemia deletions in individuals with β thalassemia trait.
Material and Methods

The study was carried out on 86 subjects of β thalassemia trait including parents and
siblings of β thalassemia major children being regularly transfused at the thalassemia
center at Guru Teg Bahadur Hospital, Delhi along with new cases of β thalassemia trait
diagnosed between September 2005 - March 2007 at the Department of Pathology,
UCMS. Most of our patients belonged to the Eastern belt of Delhi and Western U.P.
DNA was extracted from whole blood using the method of Poncz et al. The deletion
defects were detected using 2 specific oligonucleotide primers flanking the deletion
breakpoint (Figure 1). 4 primers were used in the single tube multiplex PCR. The primer
sequences and fragment size obtained are given in table 1. The primers were procured
from Microsynth, Switzerland.
The genomic DNA was amplified using a single tube multiplex long PCR method as
described earlier. In brief, the mPCR was performed in 25 µl per reaction tube, with 0.6
µmol/L each of Forward primer and Reverse mutant primer for –α3.7 and –α4.2 deletion
(Microsynth, Switzerland), 350 µmol/L of each dNTP, 2.25 mmol/L MgCl2, 7.5%
DMSO, 1.75 units of Taq polymerase (Bangalore Geneii, Bangalore, India) in 10X buffer
and 25 ng of genomic DNA in the DNA thermocycler (Perkin Elmer, USA).
The following protocol was followed:
Step Temperature Time Cycles
1. Initial denaturation 920C 2 min. 1

2. Denaturation 920C 40 sec. 10

Annealing 580C 1 min.
Extension 680C 6 min.

3. Denaturation 920C 40 sec. 20

Annealing 580C 1 min.
Extension 680C 6 min + 20 sec.
autoextension every cycle

The amplified products were examined after electrophoresis on 1.5% Agarose gel and
staining with ethidium bromide. The stained gel was then examined under UV light on a
UV transilluminator and photographs were taken using Polaroid camera, for records.
Primers used and fragment size obtained in the single tube multiplex PCR analysis of the
2 common deletions in India.
Deletion Primer Sequence Primer Indications Fragment size
per figure 1
–α3.7 5’-CCC TCC CCC Forward Primer (A) 1.76 kb
TCG CCA AGT
CCA CCC C-3’
5’-GGG GGG AGG Mutant Reverse
CCC AAG GGG Prime (B)
CAA GAA-3’
– α4.2 5’-CC TTCC TCT Forward Primer (C) 2.1 kb
CAC TTG GCC
CTG AG-3’
5’- CCC TGG GTG Mutant Reverse
TCC AGG AGC Primer (D)
AAG CC-3’

Results
α genotyping by single tube multiplex PCR revealed a total of 12 cases out of 86 (14%) β
thalassemia traits as carriers of –α3.7 deletion. 3 out of these 12 cases were homozygous
for this deletion (–α3.7 /-α3.7 ).
Agarose gel electrophoresis of 9 cases showed presence of two bands
1) 3.7 kb band indicating the presence of normal allele, and,
2) 2) 1.76 kb band indicating the presence of an amplified product derived from the
–α3.7chromosome.
Electrophoresis of 3 cases showed presence of a single band 1.76 kb in size (Fig. 2).
The band of 2.1 kb for the presence of – α4.2 deletion was not recorded in any of the
samples.
Under these experimental conditions, amplifications that would be expected for initiation
from A+B and C+D primer pairs in normal controls did not occur because the two
primers were separated by too great a distance (>5kb apart). At least one, and not more
than two bands, must appear in the gel on electrophoresis of the amplified product. If no
band is seen, then it indicates that the mPCR has failed.

Discussion
Thalassemia is a growing public health problem. According to a recent WHO study an
estimated 9,00,000 births of clinically significant thalassemia disorders are expected to
occur in the next 20 years. Worldwide, the Asian, Indian and Middle Eastern regions
account for 95% of the thalassemia births. The only approach to deal with it is the
prevention of birth of new cases. For this, the most effective strategy would be-
prevention through public education, community screening, genetic counseling and
prenatal diagnosis.
α thalassemia mutations are one of the most common mutations of man. Using DNA
analysis for screening, a prevalence of 10 – 97 % was found in different ethnic groups in
India. Thus it appears that α thalassemia is extremely common in the tribal groups of the
Indian subcontinent and indeed reaches some of the highest frequencies recorded
anywhere in the world.
The lab diagnosis of α thalassemia is important for the identification of individuals of
reproductive age who are carriers of serious hemoglobinopathy and also for the
identification of microcytic anemia to prevent unnecessary investigations to define the
etiology of anemia and avoid unwanted prolonged iron therapy.
Most of the reported studies on Indian population have used Southern Blot, RFLP and
Sequencing for the identification of the α genotype. These methods are expensive, labour
intensive, relatively complex, time consuming and require use of radioactive probes, and
therefore not practical for most of the laboratories. With the advent of Gap PCR, the
detection of α thalassemia deletions became relatively simple and cost-effective.
However, it required multiple tube amplifications (3 separate reactions) for
characterization of the 2 major, –α3.7 and – α4.2 alleles. Thus this method is not suitable for
large scale population screening. Shaji et al (2000) developed a multiplex PCR
amplification procedure using a single tube, for the detection of –α3.7 and – α4.2 alleles, the
most common cause of this disorder in India.
We have applied the latest technique of single tube multiplex PCR and found it to be a
rapid, sensitive and reliable technique. It requires a very small amount of genomic DNA
and the results are available within 5 hours, which should reduce the time and complexity
of screening for these common α thalassemia deletions. No additional inner control
indicating the success of multiplex PCR is required. Thus being rapid and cost-effective,
this multiplex PCR assay should considerably simplify carrier detection and large scale
population screening for α thalassemia deletions in at risk individuals.
Moreover it is known that the α gene number modifies the phenotype of β thalassemia by
altering the ratio of α and β chains of hemoglobin. Thus detection of –α3.7 and – α4.2
deletions in samples for prenatal diagnosis of β thalassemia major would be important
because the coexistence of these deletions in homozygous β thalassemia may ameliorate
the phenotype. Thus detection of α gene number in association with β chain defects
would provide a comprehensive prenatal diagnosis with accurate risk prediction and
appropriate genetic counseling.
This study emphasizes the efficacy of single tube multiplex PCR technique in the Indian
set up to detect defects of the α globin gene in laboratories engaged in the diagnosis of
thalassemia and prenatal screening.

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