Vol. 1(4), pp.

45-50, October 2013

Available online at http://www.accessinterjournals.org/arbs
ISSN 2350-2428
Copyright ©2013 Access International Journals

Identification of β-globin gene mutations in families and

the affected child with β thalassemia major: Importance
of prenatal diagnosis
Rubina Ghani1 , Muhammad Saboor2, Iftikhar Ahmed3, Shamim Mushtaq4 and Moinuddin5

1,3,&4
Department of Biochemistry, 2-5Department of Hematology, 4Department of Biochemistry, Zaiuddin University,
Pakistan.
1,2,,3 & 5
Baqai Medical University, 1Pathological and Molecular Laboratories, Karachi, Pakistan.

Corresponding author. E-mail: r.musavvir33@hotmail.com; Tel: 92-21-35862603, 35862612.

Accepted 20 September, 2013

β-thalassemia is an inherited disorder characterized by reduced or absent β-globin chain production. It

is the most common single genetic disorder causing a major health problem in the world. This disorder
is also common in Pakistan. Karachi is the biggest, highly populated, and cosmopolitan city with five
major ethnic groups. This study was aimed to identify the β-globin gene mutations causing β-
thalassemia in Karachi and to emphasize the importance of prenatal diagnosis. A total of 210
individuals from 70 families were screened in 11 different β-globin gene mutations by the amplification
refractory mutation system (ARMS) technique after abnormal hematological parameters. Frequency of
consanguineous marriages in parents was; first cousin 72%, second cousin 5%, distant cousin 4% and
un-related 19%. Eleven different mutations were detected. The mutations observed were IVS 1-5(GC),
619bp del, IVS 1-1 (GT), Fr 8/9 (+G)¸ Fr 41/42 (-CTTT), CD 30 (GA), CD 15 (G->A), IVS II-I (G->A), Fr 16
(-C), Cap +1 (A->G) and CD 5 (- CT). Different scientific approaches have been observed throughout the
world for screening of thalassaemia. Screening families of index child with thalassaemia has shown
promising results. Consanguinity was present in most of the parents of patients with β-thalassemia
major. Knowledge of the predominant mutation in a given ethnic group will not only help in developing
a short panel of (population-specific) primers of mutations thereby providing a cost-effective method
for prenatal diagnosis and also help the clinicians to counsel regarding blood transfusion regimen/
pregnancy termination.

Key words: β- thalassemia major, β-globin gene, prenatal diagnosis, ARMS technique.

INTRODUCTION

Thalassaemia is a hereditary disease, leading to severe affecting the hemoglobin synthesis (α or β polypeptide
and chronic hemolytic anemia that results from mutations chains of hemoglobin) (Tunaci et al., 1999; Alhaija et al.,
Adv. Res. Biol. Sci. 46
2002). β-Thalassemia is a major health problem in
Pakistan. It is the most prevalent genetically transmitted
blood disorder with a carrier rate of 5-8%; around 5000
children are diagnosed each year. In Pakistan blood is in
short supply and blood banking facilities are primitive
although developing gradually. Screened PRC is scarce
even. Due to social and economical reasons patients are
generally not able to maintain the ideal Hb level of 9-10.5
g/dl. Therefore mostly thalassaemic children are under
transfused as per international standards and their pre-
transfusion Hb is around 6-7 g/dl. As thalassemias are a
heterogeneous group of inherited disorders of
hemoglobin synthesis resulting in life-threatening anemia
and requiring regular blood transfusion for survival
(Hafeez et al., 2007).
Currently, 217 causative molecular defects have been
described so far in the β-globin gene causing β-
thalassemia (Ahmed et al., 1996). About 20 mutations
account for 90% of β-globin genes in the world and it is
noted that each ethnic population has its own unique set
of most frequent mutations. Previously, there have been
few studies investigating the spectrum of β-thalassemia
mutations in various regions and ethnic groups of
Pakistan (Khan and Riazuddin, 1998; Ansari and Shamsi,
2010).
Prenatal diagnosis for thalassaemia and related
haemoglobinopathies was initially applied over 20 years
ago through analysis at the protein level (Weatherall and
Clegg, 1981). Globally, effort have been made for
prevention of thalassemias by mass education and
various prevention strategies like, mass screening,
extended family screening of the index child, prenatal
diagnosis and termination of pregnancy and pre-marital
screening. Population screening, genetic counseling,
prenatal diagnosis and option of terminating affected
pregnancies remain the mainstay strategy to devise a
control program and investigating the underlying
molecular defects in β-thalassemia is an important
prerequisite for such programs. In Pakistan, most of the
patients live in rural areas but those who live in cities do
have access to prenatal diagnosis services which are
mostly run by charity organizations (Kanavakis et al.,
1997).
The prenatal diagnosis and termination of pregnancy is
not permissible uniformly in all religions and societies,
although increased trend is seen in modern societies and
part of the world with high literacy rate. For a successful
prevention program, support from all stakeholders
including religious scholars, electronic and print media,
gynaecologist, paediatricians, thalassaemic families,
government and non government organizations is
mandatory.
The aim of this study is to identify the frequency of
various mutations and prenatal diagnosis to provide an
accurate and rapid result as early in the pregnancy as
possible.
MATERIALS AND METHODS
The studied population included members of the five
major ethnic groups in Karachi: Punjabi, Pathan, Sindhi,
Baluchi, Immigrants (from India after the 1947 partition of
Subcontinent) and others like Mohajirs, and Memon as
well. Over a period of two years, collected venous blood
samples (in EDTA) from 210 individuals from 70
thalassemic families, (both parents with thalassemia
minor and their affected child) were collected. The ethical
clearance was obtained from the medical research ethical
committee of the Baqai Medical University. The patients
included in this study were all transfusion dependent and
had been diagnosed earlier in life as β-thalassemia major
with the help of basic hematological parameters, that is,
the red blood cell (RBC) values were obtained with an
automated cell counter (Mindray 2800, Sherizhe, China),
including complete blood counts, peripheral blood
morphology and hemoglobin electrophoresis (Helena,
Lindbergh Texas, Beaumont USA) as shown in Figure 1.
Mutation analysis for β-thalassaemia
The β- thalassaemia mutations of the carriers and the
infected child were characterized by Multiplex ARMS
PCR for the previously reported common mutations in
Pakistani populations. The mutations include: (IVS I-5 (G-
C), Fr 8-9 (+G), IVS I-1 (G-T), Fr 41-42 (-TTCT), Del 619
bp, Cd 15 (G-A), Cd 5 (-CT), Cd 30 (G-A), Fr 16(-C), IVS
II-1 (G-A) and Cap +1 (A-C) (IDT, Technology, Coralville,
Iowa, USA) (Varawalla et al., 1991).
DNAs were extracted from the whole blood samples by
using AccuPrep® Genomic DNA extraction kit (Cat #
MCD85201, Epicenter Kit USA). Finally, the extraction
containing purified DNA was used for PCR or the DNA
extraction was kept at -70°C until analysis.
After DNA extraction, polymerase chain reaction (PCR)
reactions were set up in three separate tubes for each
sample according to their base pairs. The mutant primers
used for the amplification with control primers A, B, C and
D serving as internal controls in all the PCR reaction
mixtures giving rise to 861bp fragments.
A total of 20μl final PCR reaction volume was used to
measure the PCR condition. The reaction volume was
composed of 0.5 µg of the DNA template, 10 pmol of
each of the five primers (2 control primers and 1 mutant
ARMS primer for the reaction), 2.5 unit Taq DNA
polymerase, and 0.2 mM of each deoxyribonucleotide
triphosphate (dNTP) in a solution of 10 mM Tris-HC1, 50
 Ghani et al. 47

B
Figure 1. Diagram A shows the peripheral blood smear showing microcytois poikilocytosisand macrocytosis. Diagram B shows the
Hb electrophoresis for the identification of Hb F and Hb A2 level in thalassemic families.

mM KCl and 1.2 mM MgCl2, (Promega, Madison, USA).
The thermal cycling regimen consisted of 25 cycles;
denaturation 94°C for 1 min, primer annealing at 65°C for
1 min and extension at 72°C for 1.5 min. In the final
cycle, the extension reaction was prolonged to 3 min at
72°C (Old et al., 1990).
Electrophoresis was performed using 15 μl of the PCR
products mixed with 3 μl of a loading buffer (Promega,
Madison, USA) and then loaded on a 2% agarose gel.
The gel was electrophoresed at 100 volts for 1 h and then
stained with ethidium bromide (Promega, Madison, USA).
After staining, the bands became visible under ultraviolet
(UV) light. Different mutations were characterized with
100 bp or 50 bp DNA ladders (Promega, Madison, USA).
RESULTS
By using the methods mentioned earlier, mutations were
characterized in 210. The study population included 53%
(112) males and 47% (98) females. The overall
distribution of the β-thalassemia mutations in different
ethnic groups of Pakistan settled in Karachi is
summarized in Table 1. This study provides
comprehensive data on thalassemia mutations in
different ethnic groups as well as in thalassemia parents
Adv. Res. Biol. Sci. 48

Table 1. Showing the frequencies of β thalassaemia mutations and their distribution in five major ethnic groups.

Mutation Punjab Pathan Sindhi Gujrati/ Mohajirs Balochi

(n=11) (n=14) ( n=15) (n=12) (n=15) (n=14)
IVS 1-5 6 (2.85%) 5(2.38%) 9(4.28%) 10(4.76%) 9(4.28%)
IVS 1-1 8(3.80%) 9(4.28%) 5(2.38%) 9(4.28%) 9(4.28%)
Fr 8/9 9(4.28%) 9(4.28%) 5(2.38%) 5(2.38%) 2(0.95%)
Fr41/42 8(3.80%) 2(0.95%) 2(0.95%) 5(2.38%) 5(2.38%)
Del 619 4(1.90%) 11(5.24%) 9(4.28%) 9(4.28%) 4(1.90%)
CD 5 2(0.95%) 2(0.95%) 2(0.95%) 0 2(0.95%)
CD 15 0 2(0.95%) 0 2(0.95%) 0
CD 30 0 2(0.95%) 2(0.95%) 2(0.95%) 2(0.95%)
Fr 16 2(0.95%) 0 0 2(0.95%) 2(0.95%)
IVS 11-1 2(0.95%) 0 0 2(0.95%) 2(0.95%)
Cap + 1 2(0.95%) 2(0.95%) 2(0.95%) 2(0.95%) 2(0.95%)

Figure 2a. The analysis of DNA sample with primer CD 15 mutation, and frame 41/42, in both the mixture is
identified in thalassemic families.

prevalent in Karachi.
The mutation observed were IVS 1–1(G→T), IVS 1–5
(G→C), Fr 41/42(-TCTT), Fr 8/9(+G), Del 619, Fr 16, IVS
II-I, Cd 5(-CT), Cd 30 (G-A) and Cap +1 respectively.
During this study, the mean age of the studied population
was 30.5 ± 5.82 years ranging from 22 to 45 years and
the affected child was from 8 months to 3 years.
The couples included in the study were from different
ethnic groups, that is, 14 Punjabi, 15 Pathans, 14
Balochi, 15 Gujrati/ Mohajir (Urdu speaking), and 12 were
Sindhi. The partners were first cousins in 40 cases,
distant relatives in 18 cases and unrelated in 12 cases.
Results of PCR of these couples were with their affected
child are shown in Figure 2a-b respectively.

Figure 2b. The analysis of DNA sample with primer IVS 1-1 mutation, CD 30, and del 619 in mixture is identified in thalassemic
families.
The four most common mutations; IVS 1–1 (G→T),
IVS-I-5 (G-C), Fr 8-9 (+G) and Del 619 constitute (Usman
et al., 2009). Fourteen percent of all the mutations
characterized. Fr 8-9 (+G) was the most common
mutation in Punjabis and Pathans whereas IVS 1–1
(G→T) was only common in Pathan. IVS-I-5 (G-C) was
the predominant mutation in Sindhi, Gujarati/mohajir and
Balochi. CAP+1 mutation, the only silent mutation in the
study was present in the entire ethnic group. Besides
these five common other mutations were present in less
common.
DISCUSSION
Pakistan is a country with a remarkable racial mix from a
long history of invasions and commercial interactions,
leading to considerable genetic diversity. This is
supplemented by a strong cultural preference for
consanguineous marriage has been responsible for a
relatively high prevalence of recessively inherited
disorders like β-thalassemia.
β-thalassemia is one of the commonest inherited
disorders in Pakistan. One out of every twenty individuals
carries a gene for β-thalassemia. Since the population is
Ghani et al. 49
rapidly increasing in Pakistan, therefore the birth of a
large number of new cases is expected every year
(Ahmed et al., 1996). This disease is frequently
encountered in regions of the Mediterranean basin,
Africa, South East Asia and in Indian Sub-Continent
(Ahmad et al., 2002). Information about the distribution of
different β-thalassemia mutations in various populations
is important for the establishment of comprehensive
prenatal diagnostic programs based on DNA analysis.
ARMS are a fast and reliable method for the diagnosis of
the disease.
A positive correlation between the increase in the
prevalence of β-thalassemia and the increasing tendency
for intermarriage within ethnic groups was found. Parents
of the patients are generally unaware of or fail to
understand hemoglobinopathies: if these disorders are to
be addressed, it is essential to adopt a community-based
approach. It has been reported that 49% of thalassemic
families show parental consanguinity. Mutation analysis
has determined the relationship of mutation pattern and
consanguinity and has shown that couples have an 80%
chance of a common mutation (Usman et al., 2009).
Although IVS 1-5 was the most common mutation
(40.89% of the sample), its frequency varied from 20% in
the immigrant (from India) population to 76.9% in the
Adv. Res. Biol. Sci. 50
Balochis. It should be noted that southwest Iran, which
shares border with Balochistan, also has a high
prevalence of IVS 1-5 (Ghani et al., 2002). The second
most frequent mutation was Fr 8-9, constituting 15.7% of
the allele pool. Indeed, Fr 8-9 was the most common
mutation in the Pathans (31.3%) as it was in people of
immigrates (47%). Higher prevalence of Fr 8-9 in the
Pathans has been reported previously also, (Ahmed et
al., 1996; Khan and Riazuddin, 1998) but other studies
(Ahmed et al., 1996) have commented that IVS 1-5 is the
most common mutation in southern Punjab.
The IVS–1-5 (G→C) mutation was common among
Sindhi, Gujarati/mohajir, and Balochi (13.34%). The
second most common mutation found in our study was
619 bp del. This mutation was more common in Pathans
(13.81%). This finding is not consistent with the studies of
Old et al. (1990). Previous studies of Pakistani
immigrants to the United Kingdom have shown that the
619 bp deletions are present in 56% of Sindhi and 13.9%
of native. In this study other mutations were also
identified in the entire ethnic group including CD 5 (-CT),
Fr 16, and Cap + 1 respectively. CD30 (G-A), was
common in Sindhi and Pathans, whereas IVS 11-1 was
the single mutation that was common in Punjabi’s,
Gujrati/Mohajirs (Ahmed et al., 2000).
Like all other developing countries, the Pakistani
population is growing rapidly, health delivery system is in
shambles, poverty is escalating (independent indicators
showing the absolute poverty rate of over 35%) and
resources are limited. It has now been realized that the
incidence of thalassemia major can be reduced by taking
effective measures such as identification of carriers,
genetic counseling, and prenatal diagnosis.
Preventive approaches are needed at all health care
levels throughout the country to reduce the burden of
thalassemia major in Pakistan. The four important steps
for prevention of thalassemia include: creating awareness
in the general population, extended family screening of
thalassemia patients, genetic counseling of individuals
and couples at risk and prenatal diagnosis of a suspected
or affected fetus (Qasmi, 2001).
REFERENCES
Abu Alhaija ESJ, Habbat FN, al-Omari MAO (2002).
Cephalometric measurements and facial deformities in
Ahmad S, Saleem M, Model B, Petrou M (2002).
Screening extended families for genetic hemoglobin
disorders in Pakistan. New Engl. J. Med. 347(15):
1162-1168.
Ahmed S, Petrou M, Saleem M (1996). Molecular
genetics of β-thalassemia in Pakistan: a basis for
prenatal diagnosis. Br. J. Haernatol. 94:476-482.
Ahmed S, Saleem M, Sultana N, Rashid Y, Waqar A,
Anwar M (2000). Prenatal diagnosis of β -thalassemia
in Pakistan: experience in a Muslim country. Prenat.
Diagn. 20: 378-83.
Ansari SH, Shamsi TS (2010).“Thalassaemia Prevention
Programme.” Hematology Updates 23-28.
Ghani R, Manji M.A, Ahmed N (2002).
Hemoglobinopathies Among Five Major Ethnic Groups
In Karachi, Pakistan. Southeast Asian J. Trop. Med.
Public Health 33(4): 855-861.
Hafeez M, Aslam M, Ali, Rashid Y, Jafri H (2007).
Regional and ethnic distribution of beta thalassemia
mutations and effect of consanguinity in patients
referred for prenatal diagnosis. J. Coll .Physicians
Surg. Pak. 17 (3): 144-47.
Kanavakis E, Traeger-Synodinos J, Vrettou C,
Maragoudaki E, Tzetis M, Kattamis C (1997). “Prenatal
diagnosis of the thalassaemia syndromes by rapid DNA
analytical methods.” Mol. Hum. Reprod. 3(6): 523–528.
Khan SN, Riazuddin S (1998). Molecular characterization
of β-thalassemia in Pakistan. Hemoglobin 22: 333-345.
Mujahid ul Qasmi (2001). The sharia resolutions for
complicated problems of present day (urdu). Idaratul
Quran wal uloom Islamia, Karachi. Pakistan. 245.
Old JM, Varawalla NY, Weatherall DJ (1990). Rapid
detection and prenatal diagnosis of β-thalassemia:
studies in Indian and Cypriot populations in UK. Lancet
336: 834-7.
subjects with β-thalassaemia major. Eur J orthod;24:9–
19.
Tunaci M, Tunaci A, Engin G, Ozkorkmaz B, Dinçol G,
Acunas G (1999). Imaging features of thalassemia.
Eur. Radiol. 9:1804 - 9.
Usman M, Moinuddin M, Ghani R, Usman S (2009).
Screening of Five Common Beta Thalassemia
Mutations in the Pakistani Population: A basis for
prenatal diagnosis. Sultan Qaboos Univ. Med. J. 9(3):
305–10.
Varawalla NY, Old JM, Sarkar R (1991). The spectrum of
β-thalassemia mutations on the Indian subcontinent:
the basis for prenatal diagnosis. Br. J. Hematol. 78:
242-47.
Weatherall DJ, Clegg JBb(2001). The thalassaemia
syndromes. 4th ed. Oxford: Blackwell Science;. p 237–
86.
Weatherall, DJ, Clegg JB (eds) (1981). The Thalassemia
Syndromes. 3rd edn. Blackwell Scientific Publications,
Oxford, pp. 728–737.