ABSTRACT
OBJECTIVE: To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal ß-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 ß-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with ß-thalassaemia. METHODS: 49 samples previously identified as having inherited a paternal ß-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 ß-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 ß-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders. TWEETABLE ABSTRACT: NIPT for ß-thalassaemia achieved using nested-PCR followed by relative mutation dosage.