Novel Haplotype-Based Noninvasive Prenatal Diagnosis for Recessive Single-Gene Disorders: A Proof-of-Concept Study

Accurate parental haplotype information is crucial for noninvasive prenatal diagnosis (NIPD) of recessive single-gene disorders (SGD) (NIPD-SGD). However, conventional approaches rely on complex experimental techniques or trio-based SNP linkage analysis requiring family members, which limit clinical application. Here, we present a novel direct haplotyping based NIPD approach termed DiHNIPD, utilizing single-tube long fragment read (stLFR) sequencing. First, parental genome-wide haplotypes were reconstructed by stLFR-based whole genome sequencing (WGS). Second, SNPs within and surrounding the target gene in maternal plasma were identified by WGS. Finally, fetal haplotypes were determined by implementing a parental haplotype-assisted hidden Markov model combined with the Viterbi algorithm. The DiHNIPD results were further confirmed by invasive prenatal diagnosis. This study recruited 23 couples at risk of having a fetus with SGD. DiHNIPD directly phased all parental haplotypes of the target gene and accurately deduced 23 fetal genotypes, achieving 100% concordance with diagnostic results. These results demonstrate that DiHNIPD is a sensitive, user-friendly and inexpensive strategy for NIPD-SGD. It eliminates the need for expensive devices and complex procedures and does not rely on family members. This method shows significant promise for clinical use in high-risk pregnancies without prior offspring.