Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays

Abstract

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week.

Fig 1. Allelic discrimination plots obtained from 94 total samples of genomic DNA.

The genomic DNAs extracted from healthy donors, healthy carriers and β-thalassemia patients, were analyzed with β⁰39 (A), β⁺IVSI-110 (B), β⁺IVSI-6 (C), or β⁰IVSI-1 (D) genotyping assays. For each plot, the normalized end-point fluorescence (Rn) values generated by the VIC^®-labeled probe for the normal allele and the FAM^™-labeled probe for the mutated allele are reported along the x-axis and the y-axis, respectively.

Fig 2. Amplification curves obtained by β⁰39 or β⁺IVSI-110 genotyping assays from circulating DNA extracted from maternal plasma.

Real-time PCR with β⁰39 (A) or β⁺IVSI-110 (B) genotyping assays, containing a normal probe (VIC^®, upper panels) and a mutated probe (FAM^™, lower panels) were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β⁰39 (A) or β⁺IVSI-110 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. In panel (B), #146a and #146b refer to samples collected from the same pregnant woman (number 146) at different gestational ages: 5 weeks and 10 weeks, respectively. The amplification was performed by using the StepOne^™ Real-Time PCR System (Applied Biosystems^®—Thermo Fisher Scientific).

Fig 3. Amplification curves obtained by β⁺IVSI-6 or β⁰IVSI-1 genotyping assays from circulating DNA extracted from maternal plasma.

Real-time PCR with β⁺IVSI-6 (A) or β⁰IVSI-1 (B) genotyping assays, containing a normal probe (VIC^®, upper panels) and a mutated probe (FAM^™, lower panels), were performed for circulating DNA extracted from maternal plasma where the father was a carrier of β⁺IVSI-6 (A) or β⁰IVSI-1 (B) thalassemia mutation, respectively. The plots show the ΔRn values as a function of the number of amplification cycles, while the threshold line is drawn in blue. Samples are listed according to increasing gestational ages. The amplification was performed by using the StepOne^™ Real-Time PCR System (Applied Biosystems^®—Thermo Fisher Scientific).