Introduction: Couples carrying heritable mutation/s for monogenic diseases can prevent disease transmission to their offspring by performing invasive prenatal diagnosis or PGD. Alternatively, the diagnosis of cell-free-fetal DNA in maternal blood offers a more amicable noninvasive option to at-risk couples, although practical applications of this diagnostic have yet to be described.
Aim: To assess the feasibility of performing ST-NGS in noninvasive prenatal diagnosis of monogenic disease from maternal blood.
Materials & Methods: Four pregnant couples, carrying mutation/s in the GBA gene (implicated in Gaucher Disease), were enrolled in the study. Cell-free DNA (cfDNA) was isolated from plasma of each pregnant female index (ranging from 10-31 weeks gestation). To enhance diagnostic confidence, single-nucleotide-polymorphism (SNP)-based linkage analysis was performed using mutation carrier family members, instead of sequencing the whole GBA gene. With all samples, ST-NGS, or deep-sequencing of GBA-flanking SNPs, was performed using the Truseq Custom Amplicon (Illumina) platform and in-house assay design and genotyping analysis workflows.
Results: Overall, >470 GBA-flanking SNPs (within ±200kb of GBA) were sequenced in all 4 families with a mean sequencing depth of ~2,000x per sample. This high sequencing depth facilitated the reliable detection of paternal (embryo-specific) alleles in maternal blood with as low as 1% embryo dosage. Linkage analysis identified 2 maternal and 2 paternal GBA-spanning haplotypes in all families and one haplotype from each parent was resolved in each maternal cfDNA sample.
Conclusion: The highly sensitive ST-NGS methodology appears suitable for the noninvasive prenatal diagnosis of Gaucher and likely many other monogenic disorders.
