TLA-based haplotyping & NIPD development

3. 4. 2019

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 a Monogenic NIPD (MG-NIPD) assay, 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. The MG-NIPD assay was developed using for eighteen pregnancies, focusing on the CFTR gene, the CYP21A2 gene, 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.
In summary, this MG-NIPD assay has the potential to provides families carrying a severe genetic disease with a robust and affordable methodology for non-invasive diagnosis based on a simple blood draw. We describe how this MG-NIPD assay requires standard instrumentation and has the potential to replace invasive procedures for assessment of inheritance of monogenic diseases in the future.

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