Non-invasive prenatal diagnosis of thalassemia through multiplex PCR, target capture and next-generation sequencing

Prenatal clinical detection of thalassemia involves gap-PCR and reverse dot blot (RDB) analysis of fetal DNA acquired through invasive methods. The present study aimed to develop a non-invasive prenatal diagnostic method for thalassemia based on next-generation sequencing (NGS). A total of eight families with proband children with thalassemia were recruited for the study during a subsequent pregnancy. The sequence of the thalassemia genes of the parents and proband were determined using NGS, based on a thalassemia AmpliSeq panel. Cell-free plasma DNA from pregnant women related to the aforementioned proband was analyzed using an NGS panel, based on thalassemia-associated capture probes. Heterozygous single nucleotide polymorphisms within the 10 kb regions flanking exons of the targeted thalassemia genes were acquired using probes or AmpliSeq and employed for parental haplotype construction using Trio-based panel sequencing. The fetal haplotype was deduced from the parental haplotypes and relative haplotype dosage, and subsequently validated using gap-PCR and RDB, based on invasively sampled amniotic fluid. A non-invasive prenatal diagnosis procedure from maternal plasma fetal DNA was successfully developed based on haplotype analysis. The deduced haplotypes of eight fetuses were identical to the results of invasive prenatal diagnosis procedures, with an accuracy rate of 100%. Taken together, the present study demonstrated the potential for non-invasive prenatal diagnosis of α- and β-thalassemia using NGS and haplotype-assisted analysis.