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.

Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

“…When notified about the results of the mother’s blood test, most of the mothers chose to not undergo additional checks, such as invasive prenatal diagnosis. 10-12 Currently, there are many techniques for detecting paternally inherited beta thalassemia mutations, including nextgeneration sequencing, 13 COLD-PCR and microarrays, 14 next-generation sequencing of SNPs, 15 TaqMan genotyping assay, 16 droplet digital PCR 17 and simple fetal DNA enrichment with allele-specific real-time PCRs. 18-22 The ARMS RT- PCR technique was simple, accurate, cost-effective, and able to detect paternally inherited beta thalassemia mutations directly from cell-free DNA in maternal plasma.…”

Correlation of Hematological Parameters and NESTROFT in A Routine Hematology Laboratory – A Prospective Study

“…Sanjay Piplani et al have studied the utility of NESTROFT in 150 Indian Punjabi subjects and have concluded that it is a cost-effective, valuable screening test in the rural population 3 . Giulia Breveglieri et al have studied the Taqman genotyping for detection of single nucleotide polymorphisms frequent in thalassemias 4 . They have observed in their study that these genotyping methods could be used for non-invasive prenatal diagnosis of these disorders.…”

“…However, in beta thalssemia trait, due to alteration in osmotic resistance of the affected RBC"s due to volume/surface area ratio changes [4] lysis begins at a saline concentration between 0.4-0.35% and it may not be completed even at 0.1% solution. NESTROFT is done at a saline concentration of 0.36% to ensure a high sensitivity with acceptable specificity.…”

An Early Diagnosis of Thalassemia: A Boon to a Healthy Society

“…Comprehensive screening programs highlighted recognizing carriers and offering prenatal diagnosis in pregnancies for thalassemia have been incorporated in Canada and European countries [72,73]. Different procedures have been implimented to distinguish thalassemia like genotyping assay, genotyping measur next-generation sequencing and mass spectrometry [74][75][76]. The methods are as yet testing; consequently, more investigations are expected to create and approve them and eventually lead to proficient, exact and concrete non-invasive prenatal diagnosis of thalassemia and hemoglobinopathies [77].…”