Variant haplophasing by long-read sequencing: a new approach to preimplantation genetic testing workups

Abstract

Objective: To apply long-read, third-generation sequencing as a part of a general workup strategy for performing structural rearrangement (PGT-SR) and monogenic disease (PGT-M) embryo testing.

Design: Prospective study.

Setting: In vitro fertilization unit.

Patient(s): Couples presenting for PGT-SR (n = 15) and PGT-M (n = 2).

Intervention(s): Blastocyst biopsy with molecular testing for translocation breakpoints or mutations (targets).

Main outcome measure(s): Detailed, parental-phased, single-nucleotide polymorphism (SNP) profiles around targets for selection of informative polymorphic markers to simplify and facilitate clinical preimplantation genetic testing (PGT) designs that enable discrimination between carrier and noncarrier embryos.

Result(s): High definition of chromosome breakpoints together with closely phased polymorphic markers was achieved for all 15 couples presenting for PGT-SR. Similarly, for the two couples presenting for PGT-M, tightly linked informative markers around the mutations were also simply identified. Three couples with translocations t(1;17)(q21;p13), t(3;13)(p25;q21.2), and t(12;13)(q23;q22) proceeded with PGT-SR, requesting preferential identification of noncarrier embryos for transfer. Following selection of a set of informative SNPs linked to breakpoints, we successfully performed PGT-SR tests, resulting in ongoing pregnancies with a noncarrier fetus for all couples. Similarly, with the use of tests based on informative SNPs linked to the parental mutations, one couple proceeded with PGT-M for maple syrup urine disease, resulting in an ongoing pregnancy with a disease-free fetus.

Conclusion(s): For couples contemplating clinical PGT, variant haplophasing around the target reduces the workup process by enabling rapid selection of closely linked informative markers for patient-specific test design.

Keywords: PGT-M (monogenic disease); PGT-SR (structural rearrangement); single-nucleotide polymorphism (SNP); third-generation sequencing.