Examination of X-chromosomal INDELs by ddPCR in NIPT allows female sex determination by a positive signal and fetal fraction estimation in pregnancies with female fetuses

Introduction: In families with gonosomal recessive diseases, fetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free fetal DNA in maternal plasma. When these sequences are not found, female sex of the fetus is reported. Here, we present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex by positive amplification signals.

Material and Methods: Using ddPCR we examined X-chromosomal INDELs: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to receive the population data. Population data obtained using whole genome sequencing of 100 non- related individuals of Czech origin were also used. For all examined INDELs, we tested the performance of ddPCR for mixtures 20%, 10%, 5% and 2.5% of one homozygote on the background of the opposite one. We examined the cell-free plasma DNA from 13 pregnant women bearing Y-chromosome negative fetuses. Results: We detected the minor fraction (representing the paternal X-chromosomal allele on the maternal background) in all artificial mixtures. We confirmed the presence of paternal X-chromosome in 12 out of 13 female bearing pregnancies (92.31% sensitivity).

Conclusion: We developed the ddPCR approach allowing the determination of paternal X-chromosomal alleles on maternal background for non-invasive prenatal diagnostics. After careful optimization, this approach may be potentially used for prenatal paternity exclusion and for determination of female sex in forensic samples.

Supported by grants no. VI20172020102 by Ministry of Interior of the Czech Republic, no. Progres Q25/LF1 and no. LTACH19005 of the Ministry of Education, Youth and Sport of the Czech Republic, and by the grant RVO-VFN 64165 of the Ministry of Health of the Czech Republic.