Download Molecular Analysis of Iranian Families with Sickle Cell Disease

Molecular Analysis of Iranian Families
with Sickle Cell Disease
by Maryam Ayatollahi,a Maryam Zakerinia,a,b and Mansour Haghshenasb
a
Transplant Research Center and bHematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Summary
Sickle hemoglobin is a mutant hemoglobin in which valine has been substituted for the glutamic acid
normally at the sixth amino acid of the b-globin chain. Detection of the single base pair mutation
at codon 6 of the b-globin gene is important for the prenatal diagnosis of sickle cell anemia and sickle
cell disease. Application of the polymerase chain reaction technology to detect sickle cell patients
and heterozygous carriers in a group of patients suspected for sickle cell disease was carried out. The
sample was composed of 52 normal individuals and 52 unrelated outpatients who were attending the
Hematology Research Center. All patients were interviewed. Results of their medical histories, physical
examination, and the hematological analysis were recorded. The blood samples were collected in EDTA
and genomic DNA was extracted from leukocytes. An amplified 110 base pair fragment of the b-globin
gene containing codon 6, was digested with the restriction enzyme MS II, and electerophoresed in 3 per
cent agarose. We have established the technical condition for detection of sickle cell disease using a
PCR assay. Fifteen patients having haemoglobin (Hb SS) and 37 patients in the heterozygous state
(Hb AS) were identified. We confirm that the normal controls have the Hb AA genotype. The
standardization of a highly sensitive and specific diagnostic test for sickle cell disease permitted us to
identify the normal controls, the homozygotes and heterozygotes. This amplification method is rapid,
sensitive and simple, and also has application research that is important for the prenatal diagnosis of
sickle cell disease.
Introduction
Sickle hemoglobin (Hb S), so called because of the
sickle shape it imparts to deoxygenated red cells,
is responsible for a wide spectrum of disorders that
vary with respect to the degree of anemia, the
frequency of crises, the extent of organ injury, and
the duration of survival. This disease affects over
2 million people in Nigeria with a generally severe
clinical course.1 In some parts of Africa as many
as 45 per cent of the population has sickle cell
trait, approximately 8 per cent of blacks carry the
sickle gene, and in the United States, the incidence
of sickle cell anemia is around 1 in 625 births.2 The
sickle cell gene has been reported, although with
lower frequency, in southern India, Saudi Arabia,
the Mediterranean basin, and the Middle East.2 The
Acknowledgements
The authors thank N. Rezaei and V. Moayed for their
technical assistance.
Correspondence: M. Ayatollahi, Transplant Research
Center, Shiraz University of Medical Sciences, Shiraz,
P.O. Box 71935–1119, Iran.
E-mail [email protected].
sickle cell gene has also been reported, although with
less seriousness than thalassemia, in Iran.3 Iranians
and some Indians, have different DNA structure not
encountered in Africa.4 Some genetic factors affect
the clinical severity of sickle cell disease (SCD),
including fetal hemoglobin (Hb F) level5 and b-gene
haplotypes associated with the chromosomes.6 This
haplotype is associated with a much higher Hb F
level (15–40 per cent), fewer vaso-occlusive sickle
crisis, lower complications rate, and mild clinical
condition.7 Iranian patients with SCD, on account
of their higher level of Hb F compared with AfroAmerican patients, have a less severe clinical picture.
However, they need continuous life-long medical
attention.8 Accordingly, screening for the sickle cell
gene carriers would be of great benefit.9,10
Diagnosis depends upon documentation of the
presence of sickle hemoglobin, preferably by electrophoresis.11 Rapid methods, which are less reliable
for the detection of sickle hemoglobin, include the
observation of sickling of red cells containing sickle
hemoglobin microscopically under a coverslip by
suspending the cells in a droplet of a 2 per cent
solution of sodium metabisulfite, and solubility
tests.12 However, such tests do not detect hemoglobin
C or b-thalassemia and do not reliably distinguish
ß The Author [2005]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
doi: 10.1093/tropej/fmh101 Advance Access Published on 14 April 2005
136
M. AYATOLLAHI ET AL.
between sickle trait and sickle disease and, therefore, are of limited value. With the refinement and
automation of techniques it has also been possible to
detect sickle hemoglobin accurately and economically by high-pressure liquid chromatography and by
isoelectric focusing.13
Advances in molecular techniques have allowed
the elucidation of the molecular basis of some genetic
diseases. The use of the polymerase chain reaction to
detect the sickle mutation is the method of choice for
prenatal diagnosis. This paper describes a technique
of DNA amplification in vitro and its application for
the detection of the sickle cell (Hb S) gene.
Materials and Methods
Clinical samples
Blood samples from 52 unrelated sickle cell patients
were obtained from the Hematology Research Center
of Shiraz University of Medical Sciences between
November 2001 and February 2003. SCD was
identified by a positive sickling test and confirmed
by hemoglobin electrophoresis at pH 9.2 on cellulose
acetate by the application of a 300 V current for
35 min. Fifty-two normal individuals with no signs
or symptoms of SCD were selected from the laboratory personnel. Patients and normal controls were
randomly selected from both males and females.
Hematological analysis
Complete blood count was carried out using a
counter model (Sysmex; Luton, Beds, UK). Sickle
cell hemoglobin was quantitated after elution from a
microcolumn of diethylamineethyl cellulose (DE 52)
resin as described earlier.14 Fetal hemoglobin quantitation was performed by alkaline denaturation
procedure.15
DNA extraction
One ml of cold lysis buffer, containing 0.2 g Tris, 21 g
sucrose, 0.2 g MgCl2, 2 ml Triton 100 in 200 ml of
distilled water, was added to 500 ml of EDTA-treated
peripheral blood and centrifuged. The precipitate was
washed three times and genomic DNA was extracted
by adding 100 ml of 50 mM NaOH. Subsequently the
tube was heated in a boiling water bath to solublize
the DNA.
Polymerase chain reaction (PCR)
Target DNA sequence of all samples was amplified
with the standard PCR condition,16 using the following pair of primers (from TIB, Molbiol, Berlin,
Germany), 50 -ACACAACTGTGTTCACT AGC and
50 -CAACTTCATCCACGTTCACC, that primed
amplification of an 110 base pair (bp) segment of
the b-globin gene containing codon 6. The amplified
DNA was digested with the restriction endonuclease
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Vol. 51, No. 3
MS II, which has a recognition site at codon 6 in the
normal b-globin gene.
Restriction endonuclease analysis
of the amplicon DNA
Thirty ml of the amplified product were treated with
1.5 ml of 10 U/ml of MS II restriction enzyme solution
(Boehringer, Mannheim, Germany), and digested at
37 C for 1 h in SURE/ cut buffer A in a total volume
of 35 ml. Subsequently, the digestion products were
separated according to size on a 2 per cent agarose
gel (Pharmacia, Sweden), by application of a 70 V
current for 45 min and visualized by ethidium
bromide staining under ultraviolet light.
Descriptive analysis
The MS II enzyme has a recognition site at codon 6
in the normal b-globin gene, and cleaved the normal
amplified b-globin DNA into two fragments, while
the fragment amplified from DNA of sickle cell
mutation remained uncleaved. The agarose gel
electrophoresis of the amplified DNA along with
the MS II digested products, detected the homozygote and heterozygote patients and normal
controls.
Results
A 110 bp fragment of the b-globin gene containing
codon 6, among controls and patients was amplified.
Figure 1 shows the agarose gel electrophoresis of the
amplified DNA along with the digested products
with MS II enzyme for two patients and two controls.
MS II enzyme has a recognition site at codon 6 in
the normal b-globin gene, and cleaved the normal
amplified b-globin DNA into two fragments of
54 bp and 56 bp, as an overlap band in agarose gel
electrophoresis, while the 110 bp fragment amplified from DNA of sickle cell mutation remained
uncleaved owing to a single base substitution (A!T)
at codon 6 in the mutation. Line 3 in Fig. 1 shows
the digested products of the MS II enzyme in a
homozygous patient, and line 7 represent the presence of the MS II site in normal individual. While
this site was present in all 104 chromosomes of the
normal individual, it was not found in the homozygous state of sickle cell patients. The single band
with a molecular weight (MW) of about 50 bp,
observed in line 7, is actually composed of a mixture
of two equal size products due to the fact that the
MS II site lies almost exactly in the middle of
the amplified DNA. Figure 2 shows the agarose gel
electrophoresis of the amplified DNA along with the
MS II digested products for three patients in the
homozygous state (p) and two heterozygous patients
(h). Lines 7 and 11 in Fig. 2 represent the heterozygous state with two different bands. One band
had a MW of about 110 bp corresponding to the
137
M. AYATOLLAHI ET AL.
1
−
m
2 3
− −
p p
4 5 6 7
− −
n n
Fig. 1. Line 1 is the molecular marker (50–750 bp). Lines 2 and 6 show the PCR products of the amplified DNA
from a patient in the homozygous state (p) and one normal (n) subject. Lines 3 and 7 are the enzyme-digested
products corresponding to the amplified DNA on its left line. Lines 4 and 5 show the PCR and the digested
product of a sample without DNA for negative control.
amplified DNA of sickle cell mutation which
remained uncleaved with MS II digestion, and
another band which had a mixture of two equal
size products corresponding to the normal amplified
b-globin gene. This methodology for SCD permitted
us to identify 15 patients (14.4 per cent) with sickle
cell anemia (having Hb SS) and 37 patients (35.5 per
cent) in the heterozygous state (Hb AS) (Table 1).
We confirm that the normal controls had the Hb
AA genotype.
Discussion
Sickle cell anemia is one of the most common
heritable hematologic diseases affecting humans.
Sickle hemoglobin is a mutant hemoglobin in which
valine has been substituted for the glutamic acid
normally at the sixth amino acid of the b-globin
chain. This hemoglobin polymerizes and becomes
poorly soluble when oxygen tension is lowered, and
138
red cells that contain this hemoglobin become
distorted and rigid.11 The diagnosis of sickle cell
anemia rests on the electrophoretic or chromatographic nature of hemoglobins in hemolysates prepared from the peripheral blood. However, several
relatively hemoglobin variants have an electrophoretic mobility identical to that of Hb S on cellulose
acetate.2 Although the diagnosis of SCD is straightforward, that of Hb Sb thalassemia may sometimes
be problematic. In Hb Sbþ thalassemia, there is a
preponderance of Hb S with Hb A comprising 5–30
per cent of the total. Hb Sb0 thalassemia produces an
electrophoretic pattern that is visually indistinguishable from that of sickle cell anemia, but a diagnosis
can often be made by the presence of an elevated Hb
A2 level and a decreased MCV. However, detailed
family history and DNA-based studies may be
necessary to make this distinction.2
In the last two decades, the molecular pathology
of b-thalassemia and the mutation phenotype
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M. AYATOLLAHI ET AL.
1
m
2
p
3
p
4
p
5
p
6
h
7
h
8
p
9
p
10
h
11
h
Fig. 2. Line l is the molecular marker (0.07–12.2 kbp). Lines 2, 4, 6, 8 and 10 show the PCR products of the
amplified DNA from three patients in the homozygous state (p) and two patients in the heterozygous state (h).
Lines 3, 5, 7, 9 and 11 are the enzyme-digested products corresponding to the amplified DNA on its left line.
Table 1
Identification of the sickle cell gene in patients
compared with normal controls
Studied individuals
No. tested
Heterozygous patients
Homozygous patients
Normal controls
37
15
52
Total (%)
35.5
14.4
50.0
relationships of these disorders have been largely
elucidated. This knowledge has been applied to
molecular diagnosis, carrier identification, and prenatal diagnosis and has resulted in a dramatic
reduction of the incidence of the homozygous state
in several at-risk populations.10 Detection of the
sickle cell gene by analysis of amplified DNA
sequences was done in China9 and Venezuela17 by
using the endoneuclease MS II. Hatcher, et al.18
designed an enzymatic amplification and restriction
endonuclease digestion for the detection of HbS.
In a previous study,3 we reported the frequency
of the sickle cell gene in the south of Iran. In Fars
Province, in southern Iran, with a gene frequency
Journal of Tropical Pediatrics
Vol. 51, No. 3
of around 0.01, which is one-tenth of b-thalassemia
gene frequency, the sickle cell gene is not too prevalent.3 However, this problem must have been the
most likely selective force operating in the dissemination of the sickle cell gene.19 So, screening for sickle
cell carriers would be of great benefit.17 In the present
study, we have established the technical condition for
the detection of sickle cell mutation using a PCR
assay that eliminates the need for other hematologic
analysis. This methodology permitted us to identify
the normal controls, the homozygote and heterozygote sickle cell patients. This amplification method
is specific and sensitive, thus permitting rapid,
economical diagnosis of SCD. Also, as the detection
of the single base pair mutations at codon six of the
b-globin gene is important for the prenatal diagnosis
of sickle cell gene,18 this technique has application
research that is important for the prenatal diagnosis
of sickle cell disease.
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