Preclinical workup using long-read amplicon sequencing provides families withde novopathogenic variants access to universal preimplantation genetic testing

Abstract

STUDY QUESTION Can long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents? SUMMARY ANSWER Long-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs. WHAT IS KNOWN ALREADY Universal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype. STUDY DESIGN, SIZE, DURATION This is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS To identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5–10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping. MAIN RESULTS AND THE ROLE OF CHANCE The parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT. LIMITATIONS, REASONS FOR CAUTION The main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs. WIDER IMPLICATIONS OF THE FINDINGS Targeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 ‘Methods for haplotyping single-cells’ and ZL913096-PCT/EP2014/068315-WO/2015/028576 ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’ licensed to Agilent Technologies. All other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Pseudoexon activation by deep intronic variation in GNE myopathy with thrombocytopenia

“…31 This capability, combined with its broad application range including candidate gene screening, refining complex structural breakpoints, and analyzing repeat structures, highlights LRS's potential as a transformative tool in clinical genetics. 30 The impact of different GNE variants on catalytic activity and enzymatic product transfer between domains varies significantly, underscoring the complexity in genotype-phenotype correlation. On one hand, GNE null variants have never been identified on both alleles, as these would likely be lethal.…”

“…LRS on the Oxford Nanopore platform effectively identifies pathogenic variants, including single-nucleotide variants (SNVs), copy number variations (CNVs), repeat expansions, DIVs, and methylation differences, particularly in cases where conventional genetic testing is insufficient. 30 It has proven to be an efficient and cost-effective approach for evaluating high-priority genes and regions, as well as for resolving complex cases in clinical genetics. 21 One notable advantage of LRS, as shown in P1, is its ability to determine the parental origin of inherited alleles, significantly enhancing the diagnosis and management of genetic diseases, effectively bypassing the need for parental DNA.…”

“…Comprehensive investigation of transcriptome affected by DIVs could significantly benefit the diagnostic yield for patients with rare diseases. LRS on the Oxford Nanopore platform effectively identifies pathogenic variants, including single‐nucleotide variants (SNVs), copy number variations (CNVs), repeat expansions, DIVs, and methylation differences, particularly in cases where conventional genetic testing is insufficient 30 . It has proven to be an efficient and cost‐effective approach for evaluating high‐priority genes and regions, as well as for resolving complex cases in clinical genetics 21 .…”

“…One notable advantage of LRS, as shown in P1, is its ability to determine the parental origin of inherited alleles, significantly enhancing the diagnosis and management of genetic diseases, effectively bypassing the need for parental DNA 31 . This capability, combined with its broad application range including candidate gene screening, refining complex structural breakpoints, and analyzing repeat structures, highlights LRS's potential as a transformative tool in clinical genetics 30 …”

Application and Prospects of Long Read Sequencers for Preimplantation Genetic Testing

“…Indeed, researchers have already used long-read sequencing for breakpoint-junction analysis in PGT-SR [13][14][15][16][17][18]. In the case of PGT-M, which involves embryo diagnosis for monogenic disorders, the association between pathological variants and genomic rearrangements makes long read sequencers valuable [19][20][21][22][23][24]. Reports on preclinical workups for PGT-M involve associations with SNVs and complex chromosomal structural abnormalities and the detection of deletions.…”