Non-Invasive Prenatal Detecting of Achondroplasia In Earlier Stage of Pregnancy By Droplet-Digital PCR

Background: Achondroplasia (ACH) is generally detected by abnormal prenatal ultrasound findings in the late stage of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained invasively. Most ACH cases appear to be de novo mutations with FGFR3 gene, so it is a challenge to screen ACH fetus out in the early stage of pregnancy.Objective: The aim of this study was to validate the possibility of detect fetus ACH along with non-invasive prenatal screening(NIPS) routinely in the early stage of pregnancy.Methods: 5927 cases of pregnant women undergoing NIPS were enrolled in this study. An additional 5ml of blood was collected together with NIPS blood sampling. Cell free DNA was extracted for the detecting of fetus ACH. Droplet-digital PCR(ddPCR) method based on the amplification of the two possible mutant alleles (c. 1138G>A and c. 1138G>C) of FGFR3 gene was performed to screen fetus ACH. Prenatal ultrasound and amniocentesis were then performed to confirm the positive screening result of ACH cases. The mutation sites of fetus were identified via Sanger sequencing by using amniotic fluid cells. For the screen negative cases of pregnant women, we followed up the results of prenatal diagnosis or the general conditions of the newborns.Results: One pregnant woman with fetus ACH were screened out at 22 weeks by Non-invasive prenatal detecting. Later prenatal ultrasound confirmed fetal skeletal dysplasia. Sanger sequencing confirmed de novo FGFR3 1138G>A mutant of the fetus. No ACH fetus or newborns were found in the rest detected negative cases of enrolled pregnant women.Conclusion: ddPCR technology could effectively identify de novo mutation like ACH of fetus noninvasively. We prospect the clinical application of ddPCR can expand the range of prenatal screen in the future.