Noninvasive fetal genotyping in pregnancies at risk for PKU using a comprehensive quantitative cSMART assay for PAH gene mutations: a clinical feasibility study

Abstract

Objective: To assess the diagnostic performance of a novel circulating single molecule amplification and re-sequencing technology (cSMART) method for noninvasive prenatal testing (NIPT) of Phenylketonuria (PKU). Design: Blinded NIPT analysis of pregnancies at high risk for PKU. Setting: Shanghai Xinhua Hospital and Hunan Jiahui Genetics Hospital, China. Population: Couples (n = 33) with a child diagnosed with PKU. Methods: Trio testing for pathogenic PAH mutations was performed by Sanger sequencing. In second pregnancies, invasive prenatal diagnosis (IPD) was used to determine fetal genotypes. NIPT was performed using a PAH gene-specific cSMART assay. Based on the plasma DNA mutation ratio relative to the fetal DNA fraction, fetal genotypes were assigned using a maximum-likelihood algorithm. Main outcome measures: Concordance of fetal genotyping results between IPD and NIPT, and the sensitivity and specificity of the NIPT assay. Results: Compared with gold standard IPD results, 32 of 33 fetuses (96.97%) were accurately genotyped by NIPT. The sensitivity and specificity of the NIPT assay was 100.00% (95% CI 59.04–100.00%) and 96.15% (95% CI 80.36–99.90%), respectively. Conclusions: The novel cSMART assay demonstrated high accuracy for correctly calling fetal genotypes. We propose that this test has useful clinical utility for the rapid screening of high-risk and low-risk pregnancies with a known history of PKU on one or both sides of the family. Tweetable abstract: NIPT of couples at high risk for PKU using a full-coverage cSMART PAH gene test.