A novel comprehensive non-invasive prenatal testing system for the simultaneous detection of single-gene disorders, chromosomal structural rearrangements, and aneuploidy

OBJECTIVES: Invasive prenatal diagnosis is recommended for high-risk pregnancies, but many decline due to potential complications. To date, no non-invasive prenatal testing (NIPT) simultaneously detects monogenic diseases, balanced chromosomal rearrangements (BCRs), and aneuploidies. We developed a comprehensive NIPT to detect monogenic diseases, aneuploidies, and BCRs concurrently. METHODS: We recruited eight families at risk for both monogenic disorders and BCRs. A custom capture-based single nucleotide polymorphism (SNP) panel targeting ∼60,000 loci was synthesized for target sequencing. Parental haplotypes were determined by genotyping family members. Maternal plasma DNA was sequenced and fetal haplotypes inferred by a Hidden Markov Model (HMM) to predict inheritance at targeted gene regions and rearrangement breakpoints. To validate the method's accuracy, inferred fetal genotypes were compared with amniocentesis; fetal karyotyping and a genome-wide comparison of inferred fetal genotypes were conducted in a single-blinded manner. RESULTS: All participants conceived via assisted reproductive technology. Sequencing depth averaged 200× across the 6.33 Mb target regions for cell-free DNA analysis, and the sequencing error rate ranged 0.045-0.123 % (mean 0.067 %) across all eight families. Haplotype construction succeeded for all families, enabling accurate identification of disease-causing genes and fetal karyotypes. Genotype analysis showed high accuracy for paternal and maternal informative SNPs (99.89 and 97.54 %, respectively). Fetal results from NIPT were completely concordant with invasive diagnostics. CONCLUSIONS: Our haplotype-based NIPT detects a broad spectrum of fetal genetic variations, simultaneously identifying monogenic diseases and chromosomal aberrations, and is clinically applicable as a valuable means of NIPT for the current genetic disease diagnosis.