Non-invasive prenatal testing for fetal inheritance of maternal β-thalassaemia mutations using targeted sequencing and relative mutation dosage: a feasibility study

Abstract

Objective: To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β-thalassaemia mutations in cell-free DNA. Design: Feasibility study using samples collected in a prenatal clinic. Setting: South East Asia. Population: Couples where both partners were known to be carriers of one of four common β-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β-thalassaemia. Methods: 49 samples previously identified as having inherited a paternal β-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele. Main outcome measures: Classification of the fetus as ‘unaffected’ (if the maternal wild-type allele was inherited) or ‘affected’ with β-thalassaemia (if the maternal mutant allele was inherited). Results: A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6–97.3%] and a specificity of 95.8% (95% CI 78.9–99.9%) for inheritance of maternal genotype. Conclusions: RMD for detection of inheritance of maternal β-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders. Tweetable abstract: NIPT for β-thalassaemia achieved using nested-PCR followed by relative mutation dosage.