Open Journal of Genetics, 2013, 3, 171-173 OJGen Published Online September 2013 (
Variable β-globin haplotypes in Saudi β thalassemia
Jameela Shinwari1#, Tahani Alshehri1,2#, Asma I. Tahir1, Abdullah Al Jefri3, Abdulkareem AlMomen4,
Dana Bakheet1, Mohammad AlAnazi2, Arjumand Warsy2, Nada Al Tassan1†
1Department of Genetics, Research Centre, King Faisal Specialist Hospital & Research Centre, Riyadh, KSA
2Biochemistry Department, College of Science, King Saud University, Riyadh, KSA
3Pediatric Hematology/Oncology Department, King Faisal Specialist Hospital & Research Centre, Riyadh, KSA
4Hematology Department, King Khalid University Hospital, King Saud University, Riyadh, KSA
Received 27 April 2013; revised 31 May 2013; accepted 14 June 2013
Copyright © 2013 Jameela Shinwari et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Twenty two haplotypes were generated from a pool of
60 unrelated Saudi β thalassemia major patients us-
ing previously described restriction sites in the β glo-
bin gene. Linkage disequilibrium analysis of the po-
lymorphic sites was also conducted, a few identified
haplotypes were novel while the remainder was pre-
viously reported, haplotype1222212 was the most
frequent haplotype in the study group and a strong
linkage disequilibrium between two polymorphic re-
striction sites in these β thalassemia patients was un-
Keywords: SNPs; Haplotype; Linkage Disequilibrium;
Restriction Sites
Single Nucleotide Polymorphisms (SNPs) are the most
common and easily detected type of variation that occurs
throughout the genome [1]. Models of DNA polymor-
phisms along a single chromosome are known as haplo-
types and are generated from combining the genotypes of
closely related polymorphisms that are not separated by
recombination. Naturally defined haplotypes arise as a
result of genetic variations in different populations [2],
such event appears clearly in the human β-globin gene
cluster that is extremely polymorphic and variable in
different populations, however, the polymorphic sites can
act as genetic markers used to generate chromosomal
haplotypes [3]. Generally, a mutation in the β globin
gene that occurs on a chromosome with a specific hap-
lotype is strongly linked to that haplotype [4], moreover,
as many polymorphisms are found in the β-globin gene
cluster [5], more than one β globin mutation can be asso-
ciated with one haplotype and a single mutation may be
found on several chromosome haplotypes [3]. Although
seventeen sequence polymorphisms can be detected us-
ing restriction endonucleases for haplotyping of the
β-globin gene, yet in most haplotyping studies only
seven sites are examined because the remaining poly-
morphisms are specific to a particular racial group or
linked uninformatively to sites already described. Hap-
lotype analysis tracks the history of a mutation in order
to know if there is a founder effect or a mutational hot-
spot in a specific population. The polymorphic site can
be described as 1 or (+) if a restriction enzyme cuts at the
position, and if it does not cut then the site is described
as 2 or (). Thus, the sequence of polymorphic sites can
be arranged to define the haplotype following the 5’ to 3’
order of the genes in the complex [6], the 5’ subhaplo-
type is made up of 5 restriction sites and the 3’ subhap-
lotype is made of two restriction sites covering the β-
globin gene cluster.
2.1. RFLP Genotyping of β-Globin Gene
60 Saudi β thalassemia major patients who were atten-
ding clinics at King Faisal Specialist Hospital were taken
into consideration, 10 samples were homozygous and 3
were heterozygous for the β-thalassemia mutations.
Blood samples were collected by venipuncture in EDTA
tubes and DNA was extracted from whole blood using
the DNA purification system: PuregeneTM, (Gentra sys-
*Competing Interests: None of the authors have any financial interest
related to the work presented in this manuscript and they declare no
competing interests.
#Both authors contributed equality to the manuscript.
Corresponding author.
J. Shinwari et al. / Open Journal of Genetics 3 (2013) 171-173
tem) according to the manufacturers’ instructions. Five
fragments of the DNA were amplified by PCR using
specific primers [7], and the PCR-products were sepa-
rately restricted using Hinc II, Hind III, Ava II and Hinf I
endonucleases to determine the presence or absence of
the seven restriction sites [8]. Approximately 10 - 17 µl
of PCR product were digested with 1U of the appropriate
restriction endonuclease under conditions recommended
by the manufacturer and the samples were subjected to
agarose gel electrophoresis to determine the presence or
absence of the polymorphic site. The electrophoretic
pattern was obtained for each fragment and restriction
sites were symbolized by numbers (1 - 7).
2.2. Linkage Disequilibrium and Haplotype
Haplotypes of the polymorphic sites along the β-globin
gene cluster were constructed and their frequencies were
calculated using Haploview program [9]. Linkage dise-
quilibrium analysis of the seven restriction sites was
conducted using the default settings (confidence interval
[CI] minima for strong LD: upper, 0.98; lower, 0.7;
upper CI maximum for strong recombination, 0.9;
fraction of strong LD in informative comparisons must
be at least 0.95) [9].
Twenty-two haplotypes were obtained from the study
population, Figure 1 presents these haplotypes and the
frequency of their occurrence in the Saudi patients, the
most frequent haplotype in the study group was 1222212
with a probability of 0.135, linkage disequilibrium
analysis defined a single block with linkage between
restriction sites 2 & 3 (Hind III Gγ and Hind III Aα) with
an LOD value of 5.82 and D’ value of 0.89 (89%) (Fig-
ure 2).
Discovering 22 different haplotypes in a single popula-
tion is quite significant in terms of the rate of mutational
event, however, the Saudi population is genetically het-
erogenous and therefore the genetic diversity may have
originated from gene flow resulting from population mi-
gration. In addition, since β-thalassemia is known to of-
fer a protection against malaria [9], it is believed to have
a high mutation frequency in Saudis, as malaria was in-
troduced a few thousand years ago. After exclusion of
the Ava II and Hinf I restriction sites the most common
haplotype in Saudi β-thalassemia patients was (12222)
with a frequency of 13.5% followed by 12212 with a
frequency of 11 %. Haplotypes 12222, 21121, and 21211
are known to be the most common haplotypes worldwide
[9]. In addition, it appears from comparison of the pre-
Figure 1. List of haplotypes identified in patients and their
corresponding frequencies: (a) Haplotypes in number format;
(b) Haplotypes in colored format.
sent study haplotype frequencies with populations fre-
quencies reported previously, that the frequency of hap-
lotype 12222 in the Saudi β-thalassemia patients (13.5%)
is close to the Africans (6.3%) but very low compared to
all the other populations (frequency of at least 50%);
haplotype 21211 is similar to the Southeast Asians (about
1.7%) but lower than Europeans, Indians, East Asians,
Australian Aborigines (about 20%); haplotypes 21122
(frequency 1.7%), 21111 (frequency 1.7%), 22211 (fre-
quency 5.7%) and 21222 (frequency 1.7%) are similar
to all the other population frequencies [12]. It is well
known that different ethnic groups carry their own set of
mutations and therefore specific β-thalassemia mutations
are strongly associated with specific haplotypes within
an ethnic group [13]. Linkage disequilibrium D’ value of
0.89 signifies a high LD between markers 2 and 3 indi-
cating that 89% of the chromosomes had no evidence of
historical recombination and 11% of chromosomes had
evidence of historical recombination between markers 2
and 3. Therefore either these haplotypes are too recent
leaving limited time for recombination to separate the
markers or a recent admixture between different ethnic
groups has taken place in the Saudi population and inter-
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J. Shinwari et al. / Open Journal of Genetics 3 (2013) 171-173
Copyright © 2013 SciRes.
Figure 2. Linkage disequilibrium plot (LD plot) showing a
strong linkage between marker 2 and 3 in the patient’s samples:
The numbers in the blocks represent multi-allelic D’ with
values ranging from 0 to 1(shown in percentage), Red squares
indicate statistically significant (LOD >2) allelic association
(linkage disequilibrium, LD) between pairs of markers, White
squares indicate D’ values of <1 with no statistically significant
evidence of LD, markers are numbered from 1 to 7 to the top.
breeding between groups with different alleles has dis-
torted the haplotype frequencies, causing linkage dis-
equilibrium [14].
Performing similar studies on a larger β-thalassemia
cohort would be essential for allowing the conduction of
a significant linkage between haplotypes and disease
causing mutations in the Saudi population.
This work was approved by KFSHRC IRB, Research Advisory Council
(RAC) and Research Ethics Committee (REC) (RAC# 2080012).
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