Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations

Prevention programs for beta-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different beta-globin gene mutations in Egyptian children with beta-thalassemia. Forty children with beta-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and beta-globin Strip Assay MED (TM) (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G > A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G > A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G > A (5 %), IVSII-745 C > G (5 %), IVSII-848 C > A (2.5 %), IVSI-5 G > C (2.5 %), -87 C > G(2.5 %), and cd39 C > T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G > A, and two patients were homozygous IVS I-1 G > A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting beta-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.