Noninvasive prenatal diagnosis for Duchenne muscular dystrophy based on the direct haplotype phasing.

OBJECTIVE: We aimed to investigate the validity of noninvasive prenatal diagnosis (NIPD) based on direct haplotype phasing without the proband or other family members and its feasibility for clinical application in the case of Duchenne muscular dystrophy (DMD). METHODS: Thirteen singleton-pregnancy families affected by DMD were recruited. The pathogenic variants in the pregnant females have been identified by multiplex ligation-dependent probe amplification (MLPA). We resolved maternal haplotypes for each family by performing targeted linked-read sequencing of their high molecular weight DNA, respectively. Then, we integrated the maternal haplotypes and the targeted sequencing results of maternal plasma DNA to infer the fetal haplotype and the DMD gene variant status. The fetal genotypes were further validated by using chorionic villus sampling. RESULTS: The method of directly resolving maternal haplotype through targeted linked-read sequencing was smoothly performed in 12 participated families, but one failed (F11). The predicted variant status of 12 fetuses was correct, which had been confirmed by invasive prenatal diagnosis. CONCLUSION: Direct haplotyping of NIPD based on linked-read sequencing for DMD is accurate.

Identification of copy number variants by NGS-based NIPT at low sequencing depth.

OBJECTIVE: To explore the clinical utility of detecting chromosome copy number variants (CNVs) in the fetus by noninvasive prenatal testing (NIPT) using the low-pass whole-genome sequencing. METHODS: Eight hundred and seventy-three singleton pregnancies with chromosomal microarray analysis (CMA) available between January 2017 to December 2019 and stored enough plasma sample for NIPT testing were included in this study. The CMA results show that forty-eight pregnancies with CNVs and eight hundred and twenty-five pregnancies are normal. Each pregnancy's plasma sample was blindly tested with NIPT at a depth of 0.51-1.19x for CNVs detection. The performance of the NIPT method for CNVs detection compared with the CMA method is evaluated. RESULTS: A total of fifty-two CNVs ranging from 0.1-47.3 Mb identified in forty-eight samples were identified by NIPT, of which thirty-four CNVs were consistent with CMA results. Additionally, eighteen CNVs were missed by NIPT. The overall sensitivity and specificity for the detection of CNVs were 65.38% (95% CI: 51.76%-76.89%) and 97.45% (95% CI: 96.12%-98.35%), respectively. However, for the detection of CNVs larger than 2 Mb and CNVs less than 2Mb, the sensitivities were 81.58% (95% CI: 66.27%-91.09%) and 21.43% (95% CI: 6.84%-48.32%), respectively. CONCLUSION: Our study demonstrated that the NIPT might be an alternative method for screening CNVs comparable with other studies. However, CNVs less than 2Mb in length shows poor sensitivity by NIPT. Noninvasive CNVs detection based on the NIPT method still needs more clinical validation studies and technical improvement to achieve clinically acceptable accuracy.