Myanmar Health Sciences Research Journal

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MATERIALS AND METHODS

 

Type of study and study population

A laboratory-based descriptive and analytical study was done on a total of 516 apparently healthy adults of both sexes aged between 16-45 years who were recruited from Nursing University of Yangon in 2014. Genetic origin, outcomes and prevention of beta thalassaemia disease were explained to nursing students before taking the sample. After being approved by the Institutional Ethical Review Committee, Department of Medical Research (Lower Myanmar), informed written consents were obtained from these subjects. The drug history, family history and medical history of these subjects were recorded according to the proforma in this study. Three milliliters of blood from ante-cubital vein were collected into ethylenediamine tetraacetic acid (EDTA) blood collecting tube and stored at 4ºC until the analysis.

Laboratory procedure

All fresh whole blood samples were tested for haematological parameters by Automatic Haematological Analyzer (Pantra 60) and blood film examination. Hypochromic microcytic anemia with reduced osmotic fragility samples were tested for diagnosis
of beta thalassaemia by determination of hemoglobin F% and hemoglobin A2% with G7 high performance liquid chromato-graphy (HPLC). Different types of haemo-globin were determined from whole blood samples by using isoelectric focusing gel electrophoresis (IEF). Silent beta thalas-saemia carriers were identified according to mutation or deletion of beta globin gene in chromosome 11 from blood samples of adults who had hypochromic microcytic anemia with poikilocytosis, reduced MCV result, increased HbF% and HbA2% by using molecular method of single strand conformational polymorphism polymerase chain reaction (SSCP-PCR).

Molecular method

DNA was extracted from whole blood by using the DNA extraction kit containing lysing solution (131mM NH4Cl and 0.9mM NH4HCO3), digestion solution (10mM Tris, 400mM NaCl, 2mM EDTA, 10% SDS and
3 µl of proteinase K). DNA was dissolved in 100 µl of TE buffer and stored in 4ºC refrigerator. DNA-extracted sample (15.5 µl) was mixed with 93 µl of EM solution (25mM MgCl2, 10X buffer solution and distilled water) and divided into 15 PCR tubes containing 7 µl per each tube. This solution was mixed with 3 µl of 15 different primer solutions including TE (pH 8.0), dNTP and internal primers of human growth hormone gene: 429 bp (hGHF and hGHR).


DNA amplifications were carried out in the thermocycler under following conditions; Step 1 : 95°C for 10 minutes, Step 2 : 95°C for 1 minute, 63°C for 30 seconds, 72°C
for 2 minutes (this Step 2 is repeated
10 times), Step 3: 95°C for 1 minute, 58°C for 30 seconds, 72°C for 2 minutes
(this Step 3 is repeated 20 times), Step 4 :
72°C for 10 minutes.

Amplified product was carried out to 2% agarose gel electrophoresis at 150 volt for
30 minutes and stained with ethidium bromide to detect following 15 mutant genes according to 50 bp molecular base marker.

 

Mutant genes

Combination of primers

PCR size

-28CapA>G

BARMSFLC-28CapAGR

(128 bp)

CD 5-CT

CD5-CTF-B2xRII

(306 bp)

CD 8/9+G

BARMSF-nCD8/9+GR 

(232 bp)

CD 15G>A

CD15GAF-B2xRII

(287 bp)

CD 17A>T

BARMSF-CD17ATR

(247 bp)

IVSI-1G>T

BARMSF-IVSI-1GTR

(293 bp)

CD 41/42-4del

B5UTL-41/42R

(315 bp)

CD 16-C

B5UTL-CD16-CR

(209 bp)

CD 26G>A(HbE)

B5UTL-NBEGAR

(139 bp)

IVSI-5G>C

BARMSF-IVSI-5GCR

(292 bp)

CD 35C>A

B5LUTL-CD35CAR

(308 bp)

CD 71/72+A

BInxF-n71/72+AR

(274 bp)

CD 6A>T(HbS)

BARMSF-CD6ATR

(225 bp)

-619del

619F-619R

(242 bp)

IVSII654C>T

nB2DF-n2M654R 

(258 bp)

Data collection and statistical analysis

The results obtained throughout the study were recorded and all data were analyzed by using the computer based statistical package of statistical product and service solution (SSCP) version 11.5. The results were calculated by two samples t test with equal variances (Two sample Wilcoxon rank-sum -Mann-Whitney) test in this study.

 


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