Detection of beta-thalassaemia mutations in Hong Kong Chinese using polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis

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Detection of beta-thalassaemia mutations in Hong Kong Chinese using polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis


Author: Fung, Lai-fong
Title: Detection of beta-thalassaemia mutations in Hong Kong Chinese using polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis
Degree: M.Sc.
Year: 2000
Subject: Thalassemia -- China -- Hong Kong
Mutation (Biology)
Polymerase chain reaction
Hemoglobin polymorphisms
Chinese -- China -- Hong Kong
Hong Kong Polytechnic University -- Dissertations
Department: Multi-disciplinary Studies
Dept. of Nursing and Health Sciences
Pages: ix, 70 leaves : col. ill. ; 30 cm
Language: English
Abstract: β-thalassaemia is common in South China. In Hong Kong, the carrier rate was reported to be 3.4-6.0%. Individuals with two β-thalassaemia alleles have β-thalassaemia major, a severe disorder requiring regular blood transfusion for survival. Besides, β-thalassaemia major remains a serious financial burden for patients, their families and the local health care system. However, early identification of β-thalassaemia trait can effectively reduce future mating or alert carrier couples, and hence reduce the number of newborns with β-thalassaemia major. A simple, rapid and cost-effective molecular method was developed to screen and diagnose the molecular defects of β-thalassaemia in Hong Kong Chinese. This method involved the selective amplification of three different DNA fragments (253 bp, 178 bp and 121 bp in length) from the human β-globin gene in two separate polymerase chain reactions (PCRs) so that they covered over 98% of β-thalassaemia mutations found in the local Chinese population. The three amplified products were mixed and denatured to produce single-stranded fragments and then subjected to electrophoresis (i.e. single stranded conformation polymorphism, SSCP, analysis) using 11% polyacrylamide gel (2.3% crosslinker) at 9.5-10.5C for 5 hours, followed by silver staining. The three fragments amplified from each subject were separated in a single lane. 102 normal blood samples, 104 samples from β-thalassaemia carriers and control samples with known β-thalassaemia mutations were analysed in order to define the possible normal and β-thalassaemia SSCP patterns. The SSCP patterns of the eight most common β-thalassaemia mutations found in the local population could be distinguished reliably from each other. These mutations were the deletion of CTTT at codons 41-42, C→T at IVS-II-654, A→G at nucleotide -28, A→T at codon 17, G→T at codon 43, insertion of A at codons 71-72, G→C at IVS-I-5 and C→T at nucleotide +8 in the 5' untranslated region. This technique could also detect β-thalassaemia mutation G→A at IVS-I-1, the mutation leading to haemoglobin (Hb) B (codon 26 GAG→AAG), Hb S (codon 6 GAG→GTG) and Hb C (codon 6 GAG→AAG). Moreover, it could be used to screen new β-thalassaemia mutations. The mutation leading to Hb E is present in 0.3% of the local Chinese population and reaches as high as 74% in some Thai populations. Detection of this mutation is very important because compound heterozygous inheritance of Hb E and β-thalassaemia may result in β-thalassaemia major. Besides, a new polymorphic site (IVS-II-672 A→C) within the β-globin gene was discovered. All polymorphisms occurring within the β-globin gene have to be taken into account in the SSCP diagnosis of various mutant thalassaemic alleles. The amplified β-globin gene regions covered most of the mutations found in mainland China (98-99%), Taiwan (99%) and Thailand (96%). Thus, this two-tube, single-lane technique can also be used for identifying β-thalassaemia mutations in these populations for both carrier screening and prenatal diagnosis.

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