Development of a non-invasive prenatal diagnostic assay for clinically relevant point mutations in the RTK/RAS/MAPKinase pathway

Whereas non-invasive prenatal testing (NIPT) for aneuploidies has become routine in clinical practice, NIPT for monogenic disorders is still lagging. A number of dominant conditions, including achondroplasia (FGFR3), Apert (FGFR2), Noonan (PTPN11) and Costello (HRAS) syndromes, caused by de novo point mutations in the receptor tyrosine kinase (RTK)/RAS/MAP pathway, are the most frequent spontaneous mutations in the germline with a frequency up to 1,000-fold higher than the background mutation rate. These so-called ‘selfish’ mutations arise spontaneously in spermatogonial stem cells, leading to clonal expansion and elevated mutation levels in sperm as men age. With a collective incidence as high as 1:2000 births and strong paternal age-effects, disorders associated with dominant mutations in RTK/RAS/MAPK, represent a clinically challenging group of monogenic disorders.

We have developed a targeted enrichment next-generation sequencing assay for the identification of clinically relevant de novo point mutations in cell-free fetal DNA across a panel of 45 genes. A dilution series using DNA containing up to 10 pathogenic point mutations, with variant allele frequency ranging from 1% to 10%, was created by spiking DNA from affected samples into blood DNA. Unaffected cfDNA from ten first trimester pregnancies were used as control. To estimate fetal fraction, a panel of 160 polymorphic SNP was designed. Following capture and sequencing on the Illumina NextSeq, reads were aligned to the reference genome and low frequency variants were called and annotated.

This strategy detected all 10 spiked-in variant alleles, down to 1%. The test panel was used retrospectively in a pregnancy with a positive family history of Crouzon syndrome. Analysis of cfDNA at 10 weeks’ gestation identified the FGFR2 pathogenic variant. The pathogenic variant fraction (VAF=6%) in cfDNA correlates with fetal fraction estimation of 12%. Pathogenic variants were not detected in any of the normal pregnancies tested, and all fetal variants identified were common SNPs. This prenatal diagnostic approach allows the noninvasive detection of de novo or paternally inherited point mutations and supports its potential use in prenatal screening of spontaneous monogenic disorders.