Clinical application of long‐read nanopore sequencing in a preimplantation genetic testing pre‐clinical workup to identify the junction for complex Xq chromosome rearrangement‐related disease

Abstract

Objective: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. Methods:In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications.Results: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. Conclusion:Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements. Key pointsWhat's already known about this topic? � For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for diseases with complex chromosomal structural rearrangements, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for 304 -

Application and Prospects of Long Read Sequencers for Preimplantation Genetic Testing

“…In our self-experiments, we encountered a case with complex chromosomal rearrangements for which junction detection was difficult, and even haplotyping using STR markers was challenging. In this case, an intrachromosomal insertion had occurred, and the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position [24]. If PGT-M was performed only via STR haplotyping at the original MECP2 site in this case, it may have led to misdiagnosis due to meiotic recombination.…”

“…These cases involved chromosomal structural abnormalities for which breakpoint detection was challenging using conventional cytogenetic testing, and we attempted structural analysis using long-read sequencing. In particular, we reported a case involving complex chromosome X structural abnormalities (Figure 1) [24]. We performed targeted sequencing on chromosome X using the adaptive sampling method [49,50] implemented in the desktop model GridION of the nanopore series (https://nanoporetech.com/gridion).…”

“…As the positions of junctions vary widely, the direct confirmation of the correct junction location requires the combination of cytogenetical analysis methods and is time consuming. Despite the complex chromosomal rearrangement, we were amazed that we directly detected the breakpoints and junctions in a single sequencing run using Nanopore sequencing (Figure 1A) [24]. The sequenced reads of the folded rearrangement of Junction1 were challenging to map due to the presence of segmental duplications with over 99.9% similarity.…”

“…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.…”

Long read sequencing on its way to the routine diagnostics of genetic diseases

“…In a family with three non-contiguous DMD duplications identified through standard testing, the precise breakpoint definition by LRS, showing the presence of an intact copy of the gene, contributed to the reclassification of the rearrangement as benign, with important implications for genetic counseling, especially in prenatal setting ( He et al, 2022 ). Mariya et al, 2023 used a targeted LRS approach to sequence the breakpoint junctions of a PLP1 duplication and a MECP2 duplication/triplication causing Pelizaeus‐Merzbacher disease and MECP2 duplication syndrome, respectively. In the latter case, the authors were able to map one of the MECP2 copies 45 Mb apart from the original position, avoiding a possible misinterpretation of the pathogenic allele by short tandem repeat haplotyping in pre‐implantation genetic testing, in case of recombination events occurring within this 45 Mb interval.…”