6. PGT-A METHOD ALLOWING COMBINED PGT-M FOR DETECTION OF HETEROPLASMY AND ESTIMATION OF MTDNA MUTATION LOAD IN EMBRYO BIOPSIES

Abstract

Introduction A large number of pathogenic mitochondrial DNA (mtDNA) mutations have been identified and are implicated in a variety of disorders. As mtDNA is maternally inherited, women with pathogenic mtDNA mutations are likely to have affected children, the severity of the phenotype depending on the heteroplasmy proportion. A systematic meta-analysis showed that there is a ≥ 95% chance of being unaffected at a mutant level of ≤ 18% (Hellebrekers et al, 2012). PGT-M can be used to identify embryos with mutation loads below this phenotypic threshold. The PG-Seq™ kit yields superior coverage of mtDNA and may allow combined PGT-A and PGT-M for mtDNA diseases from single biopsies. Material and methods Two cell lines, a reference and “mutant”, with mitochondrial single nucleotide variants (SNV) were selected. Six 5-cell samples from each cell line were whole genome amplified (WGA). The replicates were pooled before the cell lines were mixed in proportions ranging from 0% to 100% (mutant), in increments of 10%. Libraries were prepared for each of the pools prior to sequencing using the MiSeq instrument (Illumina). SNV sites with Results The average (± sd) mitochondrial read count for cell line samples was 5037 (± 1083). After filtering, the average (± sd) depth of coverage for the cell line samples at the SNV sites was 25.8x (± 12.5). Combining SNV data in silico to achieve a read depth typical of a standard PG-Seq™ kit 48 sample run using a single embryo biopsy (500,000 reads total), the observed SNV proportion was within 25% of the expected proportion in all cases. Combining SNV data to achieve a read depth of 5x a standard PG-Seq™ kit 48 sample run, the observed SNV proportion was within 11% of the expected proportion in all cases. This means that in any case where a heteroplasmy is detected at 7% or lower, including no detection, the mutant load should be below the phenotype threshold. Conclusions The superior amplification of mtDNA achieved by the PG-Seq™ kit may allow combined PGT-A and PGT-M for mtDNA analysis. Combining PGT-A and PGT-M in this novel way would provide a streamlined workflow over currently available workflows. While accuracy is dependent on read depth, read depth can be increased by modulating sequencing throughput or by alternate means such as using PerkinElmer s Target Sequence Enrichment protocol. Due to variable but consistent depth of coverage across the mtDNA, some sites will be more suited for analysis by this method than others.