Variable <i>β</i>-globin haplotypes in Saudi <i>β</i> thalassemia population

doi:10.4236/ojgen.2013.33019

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Open Journal of Genetics, 2013, 3, 171-173 OJGen

http://dx.doi.org/10.4236/ojgen.2013.33019 Published Online September 2013 (http://www.scirp.org/journal/ojgen/)

Variable β-globin haplotypes in Saudi β thalassemia

population*

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

Email: †[email protected]

Received 27 April 2013; revised 31 May 2013; accepted 14 June 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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-

covered.

Keywords: SNPs; Haplotype; Linkage Disequilibrium;

Restriction Sites

1. INTRODUCTION

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. METHOD

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.

OPEN ACCESS

J. Shinwari et al. / Open Journal of Genetics 3 (2013) 171-173

172

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

Construction

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].

3. RESULTS

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).

4. DISCUSSION

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-

J. Shinwari et al. / Open Journal of Genetics 3 (2013) 171-173

173

OPEN ACCESS

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.

5. ETHICAL APPROVAL

This work was approved by KFSHRC IRB, Research Advisory Council

(RAC) and Research Ethics Committee (REC) (RAC# 2080012).

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