Next-generation sequence-based preimplantation genetic testing for monogenic disease resulting from maternal mosaicism

Abstract

Background: Mosaicism poses challenges for genetic counseling and preimplantation genetic testing for monogenic disorders (PGT-M). NGS-based PGT-M has been extensively used to prevent the transmission of monogenic defects, but it has not been evaluated in the application of PGT-M resulting from mosaicism.

Methods: Four women suspected of mosaicism were confirmed by ultra-deep sequencing. Blastocyst trophectoderm cells and polar bodies were collected for whole genome amplification, followed by pathogenic variants detection and haplotype analysis based on NGS. The embryos free of the monogenic disorders were transplantable.

Results: Ultra-deep sequencing confirmed that the four women harbored somatic mosaic variants, with the proportion of variant cells at 1.12%, 9.0%, 27.60%, and 91.03%, respectively. A total of 25 blastocysts were biopsied and detected during four PGT cycles and 5 polar bodies were involved in one cycle additionally. For each couple, a wild-type embryo was successfully transplanted and confirmed by prenatal diagnosis, resulting in the birth of four healthy infants.

Conclusions: Mosaic variants could be effectively evaluated via ultra-deep sequencing, and could be prevented the transmission by PGT. Our work suggested that an NGS-based PGT approach, involving pathogenic variants detection combined with haplotype analysis, is crucial for accurate PGT-M with mosaicism.

Keywords: maternal mosaicism; monogenic disease; next-generation sequencing; preimplantation genetic testing.

FIGURE 1

The haplotypes of family members and blastocysts of cases 1–3. The yellow and green rectangles indicate the paternal haplotype, the blue rectangles indicate the maternal wild‐type haplotype, and the gray rectangles indicate the maternal disease‐causing haplotype with variant and the disease‐causing haplotype without variant. (a) Case 1, X‐linked adrenoleukodystrophy. Embryos 1, 3, 5, 8, and 9 inherited the wild‐type maternal haplotype. Embryos 2, 4, 6, and 7 inherited the disease‐causing maternal haplotype with no variant. Embryo 1 was transplanted. (b) Case 2, X‐linked Fanconi anemia. Embryos 2, 3, and 4 inherited the wild‐type maternal haplotype; embryos 1 and 5 inherited the disease‐causing haplotype without variant; embryos 6, 7, and 8 inherited the disease‐causing haplotype with the variant. Embryo 3 was transplanted. (c) Case 3, Autosomal dominant skeletal malformations. Embryos 2 and 5 inherited the wild‐type maternal haplotype; embryos 1 and 3 inherited the disease‐causing haplotype without variant; embryo 4 inherited the disease‐causing haplotype with variant. Embryo 2 was transplanted. *Embryos with disease‐causing haplotypes without variant. ET, embryo transplantation.

FIGURE 2

The haplotypes of family members, polar bodies, and blastocysts in case 4. The yellow and green rectangles indicate the paternal haplotype, the blue rectangles indicate the maternal wild‐type haplotype, and the gray rectangles indicate the maternal disease‐causing haplotype with variant and the disease‐causing haplotype without variant. The black box indicates the deletion area. (a) The haplotypes of family members. The female was a neurofibromatosis patient resulting from a de novo deletion variant. The de novo deletion occurred on the patient's maternal chromosome. (b) The haplotypes of polar bodies. PB2 of embryo 1 was degraded. None of the polar bodies carried the deletion. (c) The haplotypes of blastocysts. None of the three blastocysts carried the deletion; the heterozygous STR signals in the NF1 gene of embryos 2 and 3 were direct evidence that these embryos did not carry the deletion variant. Embryo 1 was transferred. *embryos with disease‐causing haplotypes without variant; ET, embryo transplantation; PB, polar body.