Abstract
Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β0-thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9 ± 0.1 °C were generated by normal individuals without the α-SEA deletion, 84.7 ± 0.1 °C by homozygous α-SEA deletion individuals and two melting curves at 84.7 ± 0.1 °C and 86.9 ± 0.1 °C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 ± 0.1 °C, homozygous β-FIL individuals produce amplicons with Tm at 73.6 ± 0.1 °C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6 ± 0.1 °C and 74.6 ± 0.1 °C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions.
Highlights
The β -FIL deletion is reported as the main mutation in thalassaemia patients in the indigenous populations in Malaysia
Primers for HRM analysis were optimised for different annealing temperatures, primer concentrations and polymerase chain reaction (PCR) additives
Gap-PCR combined with HRM analysis was developed in this study to detect the α -thalassaemia Southeast Asian (α -SEA) and β -FIL deletion
Summary
The β -FIL deletion is reported as the main mutation in thalassaemia patients in the indigenous populations in Malaysia. It was the single β -globin gene defect responsible for β -thalassaemia major in 20 Dusun families in Sabah[7]. Gap-PCR amplifies the deleted DNA sequence using the primers flanking the deleted region[10]. HRM analysis is a high-throughput mutation scanning method which is based on melting temperature (Tm) profiles. HRM has various additional advantages compared with other mutation scanning methods as it can detect multiple known and unknown mutations, it offers straight forward and rapid analysis. Unknown mutations detected by HRM analysis can be directly analysed and confirmed by sequencing using amplicons obtained from the same HRM assay without any delays
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