Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography

Abstract

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common β-thalassemia mutations found in Thailand. The β-globin gene was separately amplified by PCR on four different fragments covering eight most common β-thalassemia mutations including nucleotide -28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (-TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight β-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown β-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.

Phenotypic expression of Hb F in common high Hb F determinants in Thailand: roles of α‐thalassemia, 5′ δ‐globin BCL11A binding region and 3′ β‐globin enhancer

“…Although significance differences were also noted on Hb E and Hb F between the two conditions, the amount of Hb A 2 determined using the capillary electrophoresis system seems to be the most useful marker for initial differential diagnosis before being confirmed by DNA analysis. As for α‐thalassemia and β‐thalassemia , the DHPLC assays used in this study as shown in Fig. should prove useful in providing accurate genotyping platforms for these common high Hb F determinants in the region.…”

Clinical validation of a single-tube PCR and reverse dot blot assay for detection of common α-thalassaemia and β-thalassaemia in Chinese

“…[7][8][9]11 Patients in each ethnic population carry their own specific types of mutations, including a few very common ones and a variable number of rare ones. [7][8][9]11 Several molecular methods for the diagnosis of thalassaemia mutations have been described, including next-generation sequencing, 12 amplification refractory mutation system, 13,14 allele-specific oligonucleotide polymerase chain reaction, 15 denaturing high performance liquid chromatography (DHPLC), 16 reverse dot blot 17 and gap-polymerase chain reaction (Gap-PCR). 2,5,7 However, the main drawbacks of these methods are that they are expensive, labour intensive and have limited resolution.…”