P-561 Long-read amplicon guided haplotype imputation enabling comprehensive preimplantation genetic testing in families with de novo pathogenic variants

Abstract

Abstract Study question Can we use comprehensive genome-wide haplotyping PGT for couples carrying de novo mutations, given the absence of phasing references to establish the disease-associated haplotype? Summary answer Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. What is known already Preimplantation genetic testing (PGT) aims to select embryos devoid of inherited pathogenic variants. Current comprehensive genome-wide haplotyping PGT methods cannot be applied for couples where one of the partners carries a de novo mutation, as absence of affected close relatives restricts variant phasing to establish the disease-associated haplotype. For such families targeted approaches are currently used instead, thus missing genome-wide embryo analysis. Study design, size, duration To overcome the forementioned limitation, we developed a long-read amplicon guided haplotype imputation method. From January 2017 till June 2021 thirty-two couples were enrolled into the PGT program at the Centre for Human Genetics, UZ Leuven, in the context of a de novo variant causing a known monogenic disorder in one of the two partners. Participants/materials, setting, methods Genomic DNA from the partner carrying the variant (proband) and his/her parents was used for trio analysis via long-range PCR and long-read amplicon sequencing using the PacBio RSII and/or Oxford Nanopore platforms. Targeted haplotype phasing was then performed to impute the disease-associated allele. Main results and the role of chance The parental origin of the mutant allele was identified in 20 patients, resulting in the current 62.5% success rate. Most de novo mutations occurred on the paternal allele. In the remaining families, the region of interest either had no SNPs or had insufficient number of informative SNPs linked to mutation. From the successfully analyzed couples, eight have proceeded to PGT cycle and so far, three disease-free children have been born. Limitations, reasons for caution A costly family-specific work-up is required, allowing nevertheless the implementation of a generic genome-wide method at the embryo analysis stage. The sequencing cost itself is anticipated to decrease in time. To avoid non-informative results, the analysis of multiple amlpicons is recommended. Wider implications of the findings Current practices for the handling of couples with de novo variants are mostly targeted, require the combination of direct and indirect approaches and often require the multiple biopsies. Targeted amplicon long-read sequencing represents a valuable approach for PGT workup that leverages comprehensive PGT application in families with de novo mutations. Trial registration number not applicable