Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping

Abstract

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

Noninvasive Prenatal Diagnosis for Duchenne Muscular Dystrophy Based on the Direct Haplotype Phasing

“…Hui et al have successfully applied linked-read sequencing technology in combination with relative haplotype dosage analysis to noninvasive prenatal testing of families without proband 10 . Vermeulen et al combined the targeted locus amplification (TLA)-based haplotype phasing of the two parents with targeted sequencing of cell-free DNA for noninvasive prenatal testing of families without a first affected child 15 . These two-direct haplotype-based NIPD methods could accurately deduce the fetal mutation status without relying on the availability of DNA from the proband.…”

Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases

“…[ 4 ] However, cffDNA based assays for the detection of monogenic diseases, a category of diseases caused by point mutations or micro‐deletions in pathogenic genes, are limited by their complicated steps of next‐generation sequencing (NGS) and parental haplotyping. [ 5 ] Furthermore, the highly fragmented nature (≈200 bp) and low fetal fraction (with medians of 10–15%) of cell free DNA in the maternal circulation may lead to unreliable results in fetal genotyping. [ 6,7 ] Intact fetal cells for prenatal testing have attracted a great deal of attention.…”

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

“…Recently, haplotyping‐based assays have been developed, and validated, as alternative methodologies for NIPT of autosomal recessive diseases. These assays rely on deep whole‐genome or targeting sequencing of the cfDNA and the proband, applying sophisticated bioinformatics algorithms to accurately determine whether the fetus is affected or not affected by the familial disease.…”