Article Type : Research Article
Authors : Mazhar MW, Mazhar F, Mahmood J, Saif S and Javed S
Keywords : Beta-thalassemia; ARMS-PCR; Human Genome Project; mutations IVS 1-5 and FSC 8-9
Beta-thalassemia is one of the most common autosomal recessive disorders worldwide. Prevention by carrier screening and prenatal diagnosis is needed in populations with high prevalence of the disease. Keeping this in mind, this study was aimed at analyzing ?thalassemia disorder which is inherited in an autosomal recessive fashion through mutant alleles from parents to their children. Blood and fetal samples of two families were collected at MINAR hospital and sent to NIBGE. DNA was extracted from blood and CVS by phenol-chloroform method and quantified using Nanodrop. Then DNA was amplified by ARMS-PCR followed by horizontal gel-electrophoresis. Results showed the presence of two most prevalent beta-thalassemia mutations IVS 1-5 and FSC 8-9 in Pakistani families. Family A segregating ?-thalassemia was found to have IVS 1-5 mutation and parents were carrier for this mutation. Fetal sample of Family A was homozygous of the parental mutation. FSC 8-9 was the mutation found in blood samples of Family B. Parents and fetus both were carriers of this mutation. Genetic testing and prenatal diagnosis can reduce the frequency of ?-thalassemia disorders in Pakistan.
Materials and Methods
Before
the beginning of the study, formal approval was obtained from the Institutional
Review Board (IRB) of National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad, Pakistan.
Subjects study and clinical investigation
Two
families with ?-thalassemia were investigated in the present study. Families
were identified by collaborators in Multan Institute of Nuclear Medicine and
Radiotherapy (MINAR), Multan. After taking proper tests, medical history was
recorded before molecular investigation. Both families showed strong evidence
of autosomal recessive mode of inheritance and consanguinity. After approval
for the study by local ethical committee and a written consent from all
families, pedigrees were constructed from all available information using
standard method introduced by Bennett. Cyrillic (Cherwell Scientific Publishing
Ltd, Oxford, UK) software version 2.1.3 was used to draw all extensive
pedigrees. Different symbols are utilized in this software to explain
architecture of families. Males and females are represented by different
symbols i.e., males by squares and females by circles. Numbers enclosed in
these symbols represent number of individual/siblings.
Description
of families
Families
were identified from southern areas of Punjab and at least 1-3 affected
member(s) of each family was subjected to genotypic examination. Parents of
both families have consanguinity and were found as clinically healthy. Genetic
screening of thalassemic families was performed for known mutations prior to
prenatal diagnosis. CVS samples were collected at 12th week of the
pregnancy.
Family A: Blood sample of three
family members (IV: 1, IV: 2, V: 2) along with CVS sample (V: 3) were collected
from MINAR hospital for mutational screening of ?-thalassemia. This family was genotyped
for most frequent mutations of ?-thalassemia
in Pakistan. Individuals represented by IV: 1 and IV: 2 are the parents, V: 2
is sick child (proband) and V: 3 is CVS.
Family B: Blood
samples of mother and father (III: 1 and III: 2) along with CV sample (IV: 3)
was sent from MINAR hospital to HMGL. They were genotyped for 5 most prevalent
mutations of ?- thalassemia.
Samples
collection
Peripheral venous blood was taken after
the consent of the individuals or their legal guardians at MINAR, Multan after
proper diagnosis. Non-infectious 5cc syringes were used to take blood which was
then transferred into anti-coagulant EDTA vacutainers for storage. After all
the investigations, blood was transferred to Human Molecular Genetics
Laboratory (HMGL), NIBGE, Faisalabad. Genomic DNA was extracted from blood
samples using phenol chloroform method.
DNA
Extraction from CVS
DNA
is extracted from CVS samples using the following protocol. The CVS sample was
separated from normal saline and transferred into 1.5 ml micro centrifuge tube
and lysed with 500 ?l of CVS lysis buffer. It was then incubated after adding
50 ?l of proteinase K and 5.0 ?l of 10% SDS at either at 60?C for two hours or
at 37?C overnight. After incubation, 500 ?l of solution C+D was added, vortexed
briefly and centrifuged for 5 minutes at 13,000 rpm. The upper aqueous layer
was separated in new 1.5 ml micro centrifuge tube. This step was repeated
thrice in order to get highly purified DNA. Then 250?l of solution D alone was
added, centrifuged it for two minutes at 13,000 rpm, and the aqueous layer was
separated. Then 250 ?l of chloroform was added and centrifuged for 2 minutes at
13,000 rpm and the aqueous layer was separated. Then 250 ?l of 3 M Na-acetate
and 500 to 600 ?l of isopropanol (stored at - 20?C) was added and tubes were
gently inverted to precipitate DNA. It was then centrifuged for 10 minutes at
13,000 rpm. Supernatant was removed without disrupting DNA pellet, which was
then washed with 350 ?l of 70% ethanol and dried in incubator at 37?C. After
evaporation of residual ethanol, the DNA was dissolved in 30-50 ?l of TE buffer
or double distilled PCR water.
Quantification
of DNA
Concentration
of genomic DNA was determined by Thermo Scientific Nano Drop 8000 UV-VIS
Spectrophotometer. Samples were diluted 10/100 in sterile distilled water and absorbance
was measured at 260nm.
Agarose
gel electrophoresis (Horizontal)
DNA samples mixed with bromophenol blue (tracking dye)
were loaded into the wells preformed in the gels. Then electrophoresed in 0.5X
TBE buffer at 90 volts (55mA) for 45 minutes on 1% (w/v) agarose gels stained
with 4.5µl ethidium bromide (10mg/ml).The DNA fragments stained with ethidium
bromide were visualized under UV on a UV transilluminator (Uvitec, UK) and photographed.
Amplification
Refractory Mutation System (ARMS) PCR
ARMS
PCR was used for genetic screening of thalassemia families. An ARM has also
been termed allele-specific PCR or PCR amplification of specific alleles
(PASA). It is a modification of PCR and requires two oligonucleotide primers
identical in sequence except for the terminal 3’ nucleotides. One of which has its 3? terminal nucleotide complimentary to the changed sequence (Mt ARMS primer) and
other to the normal DNA sequence (N ARMS primer), both used as reverse primers
[9]. Common C was
used as a complimentary forward primer for both mutant and normal templates to
screen a specific mutation. In this system a second pair of primers was always
included in the reaction mixture to simultaneously amplify an unrelated DNA
sequence, which serves as an internal control to test that both reaction
mixture and thermal cycler is working optimally. The ARMS analysis was
performed in a reaction mixture of 20 ?l. PCR product was then visualized on
1.8% agarose gel. Amplified PCR products were resolved on 1.8-2% w/v agarose
gel. Samples mixed with bromophenol blue were loaded onto gel and
electrophoresis was performed at 90 volts (55mA) for 45 minutes in 0.5X TBE
(running buffer) filled electrophoresis tank.
Mutational analysis
Two
families of ?-thalassemia i.e. Family A and Family B were screened for mutation
screening via an improvised PCR called as amplification refractory mutation
system (ARMS). Parents of both families were first diagnosed for thalassemia
minor and then screened for most frequent mutations in Pakistani population in
order of the prevalence of these mutations. First trimester prenatal diagnosis
was carried out by chorionic villus sample DNA to screen pregnancy at risk of
?-thalassemia.
Family A: Proband of this family is receiving regular blood transfusion every month. After DNA was extracted from the blood of these samples, DNA was quantified using Thermo Scientific Nano Drop 8000 UV-VIS Spectrophotometer. Then it was followed by ARMS PCR. ARMS PCR was run for the most frequent mutation IVS-I-5 (G-C) in HBB. Homozygous (affected), heterozygous (carrier) and negative controls were also loaded along with family samples for confirmation of results. Results were checked on 1.8% agarose gel electrophoresis by comparing intensity and sharpness of the DNA bands with control samples (Figure 1-5).
Both
parents were carrier (heterozygous) while the sick child in the family
(proband) was homozygous (inherit both mutant alleles, one from each parent)
for this mutation. CVS specimen was checked for parental mutations. The
ARMS-PCR results showed that fetus is homozygous for IVS1-5 mutation (IVS
1-5/IVS1-5).
Family B: The
genomic DNA of all the extracted samples was of good quality (Table 1-3).
Then
Family B was first screened by ARMS PCR for IVS-I-5. Results showed that family
is negative for this mutation. These samples were then screened for second most
prevalent mutation FSC 8-9, for which they were found carriers. Chorionic
villus sample was idenfigutified as carrier for this particular mutation.