Droplet-based digital PCR for non-invasive prenatal genetic diagnosis of α and β-thalassemia

Non-invasive prenatal diagnosis (NIPD) of isolated cell-free DNA from maternal plasma has been applied to detect monogenic diseases in the fetus. Droplet digital PCR (ddPCR) is a sensitive and quantitative technique for NIPD. In the present study, the development and evaluation of ddPCR-based assays for common α and β-thalassemia variants amongst the Asian population was described; specifically, Southeast Asian (SEA) deletion, HbE, and 41/42 (-CTTT). SEA is caused by deletion of a 20 kb region surrounding the α-globin gene, whilst HbE and 41/42 (-CTTT) are caused by point mutations on the β-globin gene. Cell-free DNA samples from 46 singleton pregnant women who were carriers of these mutations were isolated and quantified using ddPCR with specially designed probes for each target allele. Allelic copy number calculation and likelihood ratio tests were used to classify fetal genotypes. Classification performances were evaluated against ground truth fetal genotypes obtained from conventional amniocentesis. Copy number variation analysis of SEA deletion accurately classified fetal genotypes in 20 out of 22 cases with an area under the receiver operating characteristic curve of 0.98 for detecting Hb Bart's hydrops fetalis. For HbE cases, 10 out of 16 samples were correctly classified, and three were inconclusive. For 41/42 (-CTTT) cases, 2 out of 8 were correctly classified, and four were inconclusive. The correct genotype was not rejected in any inconclusive case and may be resolved with additional ddPCR experiments. These results indicate that ddPCR-based analysis of maternal plasma can become an accurate and effective NIPD for SEA deletion α-(0) thalassemia. Although the performance of ddPCR on HbE and 41/42 (-CTTT) mutations were not sufficient for clinical application, these results may serve as a foundation for future works in this field.