Indian Journal of Human Genetics
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Year : 2005  |  Volume : 11  |  Issue : 2  |  Page : 80-83

Molecular characterization of mutations causing β -thalassemia in Faisalabad Pakistan using the amplification refractory mutation system (ARMS-PCR)

Health Biotechnology Division, National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan

Correspondence Address:
Shahid M Baig
Human Molecular Genetics Labortory,Health Biotechnology Division, NIBGE, P.O. Box 577, FSD
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0971-6866.16806

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BACKGROUND : Faisalabad is the third biggest city of Pakistan. Majority of the population is Punjabi while other ethnic groups are in minority. AIMS : The present study was undertaken to find the mutations causing β -thalassemia in Faisalabad Pakistan. MATERIALS AND METHODS : A total of 285 β -globin alleles from 143 unrelated families having at least one transfusion-dependent child were analyzed by using amplification refractory mutation system (ARMS-PCR). RESULTS : FSC-8/9 (+G) and IVS-I-5 (G ®C) were the most common mutations. The allele frequency for FSC-8/9 (+G) was 38.59% while frequency for IVS-I-5 (G ®C) was 37.89%. The high frequency (76.48%) of IVS-I-5 (G ®C) and FSC-8/9 (+G) on various alleles provides a strong evidence of intermarriages. CONCLUSIONS : By using ARMS-PCR, the mutations were successfully characterized in 95.79% of subjects, while 4.21% remain to be characterized. This study will facilitate the implementations of genetic counseling and prenatal diagnosis in the population of Faisalabad.

Keywords: ARMS-PCR; β -thalassemia; mutations detection; Faisalabad; mutations; Pakistan.

How to cite this article:
Baig SM, Rabbi F, Hameed U, Qureshi J A, Mahmood Z, Bokhari S H, Kiani A, Hassan H, Baig J M, Azhar A, Zaman T. Molecular characterization of mutations causing β -thalassemia in Faisalabad Pakistan using the amplification refractory mutation system (ARMS-PCR). Indian J Hum Genet 2005;11:80-3

How to cite this URL:
Baig SM, Rabbi F, Hameed U, Qureshi J A, Mahmood Z, Bokhari S H, Kiani A, Hassan H, Baig J M, Azhar A, Zaman T. Molecular characterization of mutations causing β -thalassemia in Faisalabad Pakistan using the amplification refractory mutation system (ARMS-PCR). Indian J Hum Genet [serial online] 2005 [cited 2017 Mar 30];11:80-3. Available from:

Thalassemia is a single gene disorder of hemoglobin (Hb), characterized by the deficient or abolished synthesis of one or more of the globin chains of Hb. β -Thalassemia is one of the major monogenic single gene disorders in the world population and it was the first disease studied by using the techniques of molecular biology. Indian subcontinent and South-East Asia have the highest prevalence of b-thalassemia and comprise the so-called ′thalassemia belt.′[1],[2] At molecular level, b-thalassemia represents a great heterogeneity as more than 380 mutations have been identified for the β -globin gene responsible for this disease (OMIM, 2005). The frequency and spectrum of these mutations vary among different populations. Immigration plays a major role in both the distribution and the extent of mutation variations within each country.[3] β -Thalassemia is one of the most common genetic disorders in Pakistan. The carrier rate in different regions of Pakistan varies from 1.4 to 8.0% with an average of 5.0%.[4] In order to control b-thalassemia, a comprehensive study dealing with molecular diagnosis of mutation causing this disease is needed for carrier detection and establishment of prenatal diagnostic program.[5] Preventive programs for b-thalassemia consisting of genetic counseling, carrier detection, and prenatal diagnostic are the effective approaches.[6] The introduction of chorionic villus sampling (CVS) along with polymerase chain reaction- (PCR) based technique has made the prenatal diagnosis rapid and reliable at early stage of pregnancy.[6] These mutations can be detected by amplification refractory mutation system (ARMS). However, for characterization of rare mutations of β -thalassemia gene sequencing and single strand conformation polymorphism techniques are reliable tools.[7]

  Materials and methods Top

A total of 285 alleles from 143 unrelated families, were analyzed for 17 b-globin gene mutations reported in Pakistan. These subjects were identified at Health Biotechnology Division NIBGE, Faisalabad with collaboration of Ali Zaib Blood Transfusion Center, Chiniot blood bank and Allied Hospital, Faisalabad.

DNA extraction

Blood samples (5-10 ml) from 143 families having at least one transfusion-dependent child with thalassemia major/intermedia were collected in vacutainer with EDTA as anticoagulant and kept at -20°C until DNA extraction. The DNA from leukocytes was extracted by NaCl method.[8] While isolation of DNA from CVS was done following the protocol of Jackson et al.[9]

The isolated DNA was dissolved in 0.5-1 ml TE buffer (20 mM Tris pH 8.0, 1 mM EDTA). The isolated DNA was evaluated by agarose gel electrophoresis after staining with ethidium bromide and exposure to UV light. Impure DNA samples were re-extracted with phenol/chloroform method.

Characterization of mutations by ARMS-PCR

ARMS-PCR allows the characterization of point mutations directly by the presence or absence of amplification using allele specific primers. For the diagnosis of specific point mutation a pair of allele-specific primers one of which has its 3′ terminal nucleotide complementary to the point mutation (Mt ARMS primer) and other to the normal DNA sequence (N ARMS primer) was used.[10],[11] Each DNA sample was screened for the 17 most common mutations reported in Pakistan. The samples uncharacterized for 17 most common mutations will be sequenced. The ARMS analysis was performed in a reaction mixture of 20 ml containing 1X PCR buffer, 50 pmoles of each of four primers, 0.5-1 mg of genomic DNA, 0.25 mM of dNTPs, 1.5 mM MgCl2, and 0.5-1 U of Taq polymerase. The thermal cycling consisted of 33 cycles of denaturation at 94°C for 45 s and primer annealing and extension at 67°C for 1.5 min. In the final cycle, the extension was prolonged for 6 min. After amplification the entire product was mixed with 1X loading dye and electrophoresed on a 1.8% agarose gel containing ethidium bromide for 20-60 min at 100 V, visualized and photographed under UV light.

  Results Top

In this study, a total of 285 β -globin alleles from 143 unrelated families having at least one transfusion-dependent child were analyzed. By using rapid DNA analytical techniques like, PCR and ARMS-PCR we have characterized 273 (95.79%) alleles out of 285 for 17 mutations, i.e., FSC-8/9 (+G), IVS-I-5 (G ® C), Cd 41/42 (-CTTT), 619 bp del, IVS-II-1 (G ® A), IVS-II-848 (C ® A), IVS-I-I (G ® T), IVS-I-1 (G ® A), Cd 15 (G ® A), Cd 16 (-C), Cd 26 (G ® A), Cd 30 (G ® C), Cd 30 (G ® A), Cd 39 (C ® T), -88 (C ® T), Cap+1 (A ® C) and Initiation Cd (T ® C). The percentage prevalence of these mutations shows that FSC-8-9 (+G) is the predominant mutation (38.59%) while the second common mutation in these chromosomes is IVS-I-5 (G ® C) with prevalence of 37.89% and third common mutation is the Cd 41/42 (-CTTT) (9.12%). The 619 bp deletion at the 3′ end of the b-gene is common and even that is restricted to India and Pakistan, where it accounts for approximately one third of the β -thalassemia alleles.[12],[13] Deletion mutation, 619 bp, was found only in 3.15% chromosomes in this study.

The pattern of b-thalassemia mutations in Faisalabad is given in [Table - 1]. In this study, two prenatal diagnoses were carried out by using CVS at 12-13 weeks. The ARMS-PCR method was used for prenatal diagnosis of β -thalassemia mutations in families at risk. The mutations had already been characterized in our screening program prior to receipt of fetal sample for families at risk. Results were delivered in 1-3 days. In prenatal diagnoses, one fetus was heterozygous (carrier/trait) and pregnancy was continued. While the other pregnancy was terminated in first trimester as fetus was diagnosed homozygote for parental mutations. In both cases the parents were first-degree cousins and carriers for the same mutation, i.e., IVS-I-5 (G ®C) and 619 del.

  Discussion Top

The information on distribution pattern of different b-thalassemia mutations in various populations is important for the establishment of comprehensive prenatal diagnosis programs based on DNA analysis. ARMS is fast and reliable method. In this study, 285 alleles were analyzed for b-thalassemia mutations in Faisalabad. FSC-8/9 (+G) is diagnosed as the most common mutation in Faisalabad (38.59%). However, our group and others have already reported this mutation with different frequency rates in various regions of Pakistan as 44% in North West Pakistan, 41% in Northern areas of Pakistan, 36% in Bahawalpur, and 4.5% in Karachi. [4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14]

The second most common mutation is IVS-I-5 (G ®C) (37.89%). High frequency of FSC-8/9 and IVS-I-5 mutations suggests that these may be the oldest b-thalassemia mutations in the Indian subcontinent.[7] Though IVS-I-5 (G ®C) mutation is the most prevalent mutation of b-globin gene almost all over Pakistan and India, the overall distribution of mutations differs radically between different regions of the country. [4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14]

The third most common mutation in this region is CD41/42 (-CTTT) that constitutes about 9.12%. In Pakistan, 20 mutations are reported but most of the mutations are less common or rare and frequency differs in different regions.[4]

In Faisalabad region, only ten mutations were detected: that is, FSC-8/9 (+G), IVS-I-5 (G ® C), Cd 41/42 (-CTTT), 619 bp del, IVS-II-848 (C ® A), IVSI-1 (G ® A), IVS-II-1 (G ® A), Cd 15 (G ®A), IVS-I-1 (G ® T) and Cap+1 (A ® C). The present results are particularly from Faisalabad while previous data and reports from other groups working in Pakistan did not represent the mutations pattern in Faisalabad.[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15] By using 16 ARMS-PCR primer sets and a direct PCR method for the detection of 619 bp deletion mutation, we have characterized 95.79% of the alleles in this study. In a previous study by Bukhari[16] 40.08% true homozygotes children were reported. In this study 70% patients are found to be true homozygotes, i.e., having same genotype. The rate of first cousin marriages among the parents of affected children is 63%. Total consanguinity rate including second cousin relationships and beyond is 81% while 19% are unrelated [Figure - 1]. This data confirms the relationship of consanguinity and high rate of true homozygousity of mutations in population of Faisalabad. There are only few centers, which offer prenatal diagnosis of b-thalassemia in Pakistan. In the vicinity of Faisalabad, there is no facility of prenatal diagnosis. Therefore considering this matter of great importance, we established prenatal diagnosis services at NIBGE and offered prenatal diagnosis to couples at risk.

  Acknowledgment Top

We are thankful to Doctors and staff of Ali Zaib Blood Transfusion Center, Allied Hospital/Punjab Medical College and Chiniot Blood Bank, Faisalabad for providing the blood samples of thalassemic children and their families.

  References Top

1.Cao A, Luisella S, Galanello R, Rosatelli MC. Molecular diagnosis and carrier screening for β -thalassemia. JAMA 1997;278:1273-7.  Back to cited text no. 1    
2.Weatherall DJ, Clegg JB. The β -thalassemia syndromes. 4th Edn. Oxford: Blackwell Scientific publication; 2001. p. 635-87.  Back to cited text no. 2    
3.Gupta A, Hattori Y, Agarwal S. Initiation codon mutation in an Asian Indian Family. Am J Hematol 2002;71:134-6.  Back to cited text no. 3    
4.Ahmed S, Saleem M, Sultana N, Raashid Y, Waqar A, Anwar M, et al. Prenatal diagnosis of β -thalassemia in Pakistan: experience in a Muslim country. Prenat Diagn 2000;20:378-83.  Back to cited text no. 4    
5.Khan SN, Riazuddin S, Galanello R. Identification of three rare β -thalassemia mutations in Pakistan. Hemoglobin 2000;24:15-22.  Back to cited text no. 5    
6.Tuzmen S, Tadmouri GO, Ozer A, Baig SM, Ozcelik H, Basaran S, et al. Prenatal diagnosis of β -thalassemia and sickle cell anemia in Turkey. Prenat Diag 1996;16:252-8.  Back to cited text no. 6    
7.Agarwal S, Hattori Y, Agarwal SS. Rare beta β -thalassemia mutations in Asian Indians. Prenat Diagn 2000;20:986-91.  Back to cited text no. 7    
8.Miller M, Dykes DD, Poesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acids Res 1988;16:215.  Back to cited text no. 8    
9.Jackson DP, Hayden JD, Quirke P. Extraction of nucleic acid from fresh and archival material. In PCR A Practical Approach, McPherson MJ, Quirke P, Taylor GR (eds). Oxford University Press: New York; 1991. p. 30-2.  Back to cited text no. 9    
10.Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucl Acids Res 1989;17:2503-16.  Back to cited text no. 10    
11.Old JM, Varawalla NY, Weatherall DJ. Rapid detection and prenatal diagnosis of b-thalassemia: studies in Indian and Cypriot populations in the U.K. Lancet 1990;336:834-7.  Back to cited text no. 11    
12.Varawalla NY, Old JM, Sarker R, Venkatesan R, Weatherall DJ. The spectrum of β -thalassemia mutations on the Indian subcontinent: the basis for prenatal diagnosis. Br J Hematol 1991;78:242-7.  Back to cited text no. 12    
13.Baysal E, Sharma S, Wong SC, Jogessar VB, Huisman TH. Distribution of β -thalassemia mutations in three Asian Indian populations with distant geographic locations. Hemoglobin 1994;18:201-9.  Back to cited text no. 13    
14.Baig SM, Rabbi F, Yaqoob N, Baig S, Khan MM, Shamsi ST, et al. Molecular study and prenatal diagnosis of β -thalassemia in Pakistan by ARMS-PCR. First Scientific Symposium of The Children Hospital Lahore held on December 3-4. 1999.  Back to cited text no. 14    
15.Khan SN, Zafar AU, Riazuddin S. Molecular genetic diagnosis of β -thalassemia in Pakistan. J Pak Med Assoc 1995 ;45: 66-69.  Back to cited text no. 15    
16.Bukhari SH. Molecular spectrum and characterization of β -thalassemia in Pakistan. M.Phil. Thesis, submitted to Quaid-i-Azam University, Islamabad, Pakistan. 1996.Pp: 57-59.  Back to cited text no. 16    


[Figure - 1]


[Table - 1]

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