P-708 Establishment of linkage phase, using Oxford Nanopore Technologies, for Preimplantation Genetic Testing of Coffin-Lowry syndrome with a de novo RPS6KA3 mutation
Authors: Wen X , Fang X , Du J , Huo J , Li J , Liu N , Lin X 
Published: June 2023

Study question

There was a challenge to establishing haplotype for PGT in the family with de novo mutation because of the lack of information from affected members.

Summary answer

The ONT platform combined with MARSALA method can be used to perform PGT for DNM patients without the need for other samples as a reference.

What is known already

The proband was a 31-year-old female who was born with mental retardation and language delay. On physical examination, she was found to have distinctive Coffin-Lowry Syndrome features, such as distance between the eyes and low nasal bridge. However, the proband’s parents did not show any abnormalities. The couple underwent genetic counseling and whole-exome sequencing (WES). The results showed that a frameshift mutation (c.1496delG, p.Gly499Valfs*25) was identified in the RPS6KA3 gene, with a true mutation in the proband but no mutations in the parents.

Study design, size, duration

A couple requested genetic counseling at the reproductive center of Zhongshan Boai Hospital and underwent whole-exome sequencing (WES) in 2019. The proband then underwent PGT with Oxford Nanopore Technologies (ONT) and the MARSALA platform to block the transmission of the pathogenic mutation to her offspring in 2022.

Participants/materials, setting, methods

The proband underwent PGT to block the transmission of the pathogenic mutation to her offspring.We used long-read sequencing on the ONT platform to directly detect the mutation and nearby SNPs for construction of the haplotype in PGT. The MARSALA method was used to detect both the SNP-based haplotype and chromosome copy number variations in each blastocyst. Finally, a normal embryo was selected by comparison to the haplotype of the proband and transferred into the uterus.

Main results and the role of chance

Using WES, we found the novel, heterozygous, pathogenic c.1496delG (p.Gly499Valfs*25) mutation of the RPS6KA3 gene in the proband. The SNP-based haplotype that was linked to the pathogenic mutation site was successfully established in the proband, without the need for other family members to be tested with ONT. Eight blastocysts were biopsied to perform PGT and were assessed with a haplotype linkage analysis (30 SNP sites selected), to give results that were consistent with direct mutation detection using Sanger sequencing. The results of PGT showed that three of the eight blastocysts were normal, without the DNM. Moreover, the patient had a successful pregnancy, after transfer of a normal blastocyst into the uterus, and delivered a healthy baby.

Limitations, reasons for caution

This strategy has only been applied to one case and more cases with de novo mutaition are needed to prove the feasibility of this strategy for PGT-M.

Wider implications of the findings

This study is the first report of a de novo RPS6KA3 c.1496delG mutation in patient with CLS, which can expands the RPS6KA3 mutation spectrum and also provides a new strategy based on ONT and MARSALA to perform PGT-M with DNMs without the need for a pedigree sample as a reference.

Trial registration number

Not applicable