Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing

Abstract

Purpose: Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M).

Method: PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS.

Results: Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1-6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results.

Conclusion: These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS.

Trial registration number: N-20180001.

Keywords: PGT-M; Prenatal testing; STR markers; cbNIPT.

Fig. 1

Results from case 1. a Flowchart describing the setup and STR markers used for PGT-M and cbNIPT as well as the results and conclusions from PGT, cbNIPT, and CVS. b STR profiles from cbNIPT including paternal and maternal profiles. Insert in the upper right corner details the affected gene and the locations of the STR markers used. Affected alleles are written in red, unaffected in green (the affected parent) or blue (the unaffected parent). Alleles of indeterminable origin are written in black. A couple (29 and 32 years old) seeking PGT due to the female partner being affected by neurofibromatosis type I caused by a deletion (c.7907+4_7del) in the NF1 gene (a). Two STR markers, one fully informative located 1.7 Mb upstream of the NF1 gene (STR 1, NF1-4, see supplementary materials and methods for genomic locations and sequence of self-annotated STR markers) and one semi-informative located 1.3 Mb downstream of the NF1 gene (STR 2, D17S1880), were identified (a insert). Direct mutation detection (by fragment analysis because the mutation is a deletion) coupled with STR analysis was performed on DNA from lysed biopsied trophectoderm cells. An unaffected blastocyst was transferred resulting in pregnancy (a). CVS and blood sampling were performed in gestational week 10+5. Two potential fetal cell samples were isolated from the maternal blood sample (C1-S1 and C1-S2). C1-S2 was classified as inconclusive due to the absence of the paternal allele. C1-S2 was classified as an unaffected fetal cell with the same profile as the transferred embryo. Combined, cbNIPT confirmed the transfer of an unaffected embryo, which was also confirmed by CVS analysis (a). bp, base pair; cbNIPT, cell-based non-invasive prenatal testing; CVS, chorionic villous sampling; Mb, mega bases; PGT, preimplantation genetic testing; PGT-M, PGT for monogenic disorders; STR, short tandem, Cx-Sy, case x sample y

Fig. 2

Piecharts of cbNIPT outcomes from the eight cases. a Proportion of cell samples succesfully tested. b Origin of successfully tested cell samples. c Proportion of fetal cell samples with an “unaffected,” “conditionally unaffected,” or “affected” test result. Number, percentage, range, and median are indicated on each slice of the pie charts