Prenatal diagnosis of βthalassaemia by reverse dot‐blot hybridization

Abstract

Thalassaemia is the most common genetic disease and is a public health problem of Thailand. Prevention and control of beta-thalassaemia diseases need accurate diagnosis of carriers and proper genetic counselling. Prenatal diagnosis is needed to prevent birth of the thalassaemic offspring in the couple at risk. This can be performed in the first trimester of pregnancy by DNA analysis using the polymerase chain reaction (PCR). Since there are more than 20 mutations causing beta-thalassaemia in Thailand, the point mutation detection by reverse dot-blot allele-specific oligonucleotide (ASO) hybridization was developed using two sets of ASO probes. The first battery of ASO probes has been designed to detect 10 common beta-globin gene mutations including codon 26, G->A (Hb E): codons 41/42, -TCTT; codon 17, A->T; IVS 2 nt 654, C->T; IVS 1 nt 1, G->T; IVS 1 nt 5. G->C; codon 19, A->G (Hb Malay); codon 35, C->A; codons 71/72, +A and -28 ATA, A->G. The second set of ASO probes detect 14 uncommon beta-thalassaemia mutations. We applied this reverse dot-blot hybridization technique to perform prenatal diagnosis in 105 pregnancies at risk of having severe beta-thalassaemia diseases. 36 fetuses (34 per cent) were found to be affected with homozygous beta-thalassaemia or beta-thalassaemia/Hb E disease in which one was twin pregnancy. The others included 31 fetuses with heterozygous beta-thalassaemia, 22 heterozygous Hb E, 1 homozygous Hb E and 16 normal fetuses. The common set of ASO probes detected about 95 per cent of cases which suggests that prenatal diagnosis for beta-thalassaemia disease can be easily carried out by this approach.

ASSOCIATION OF Hb HOPE [β136(H14)Gly → Asp] AND Hb H DISEASE

“…The results showed that F.K. This was confirmed by reverse dot-blot hybridization of the PCR product with the oligonucleotide probe specific for the mutation (15). had the a-thal-1 (À À SEA ) and Hb CS genes ( Table 1).…”

Molecular analysis of β‐thalassemia in South Vietnam

“…The ␤-thalassemia mutations were characterized by polymerase chain reaction (PCR) and reverse dot-blot hybridization with the common allele-specific oligonucleotide (ASO) probes as previously described [14]. As it has been known that the frameshift mutation at codon 95 (+A) is also common among ␤-thalassemia patients in North Vietnam, the amplification refractory mutation system (ARMS) was used.…”

Identification of β‐globin gene mutations in Thailand using an automated fluorescence‐based DNA sequencer

“…β‐Globin gene fragments were amplified using 5′ biotinylated oligonucleotide primers and hybridized to 17 membrane‐bound ASO probes for −31 (A‐G), −30 (T‐C), −28 (A‐G), codons 8/9 (+G), codon 15 (G‐A), codon 17 (A‐T), codon 19 (A‐G), codon 26 (G‐A), codons 27/28 (+C), IVS 1:1 (G‐T), IVS 1:5 (G‐C), codon 35 (C‐A), codon 41 (−C), codons 41/42 (−CTTT), codon 43 (G‐T), codons 71/72 (+A) and IVS 2:654 (C‐T) mutation as previously described (Winichagoon et al. , 1999).…”

“…A variety of techniques based on polymerase chain reaction (PCR) amplification of DNA have been developed to identify globin gene mutations, such as dot‐blot hybridization, reverse dot‐blot hybridization (RDB), allele specific PCR (ARMS) and restriction enzyme analysis (Fucharoen et al. , 1995; Winichagoon et al. , 1999).…”