Background and aims: Certain chromosomal structural variations (SVs) in biological parents can lead to recurrent spontaneous abortions (RSAs). Unequal crossing over during meiosis can result in the unbalanced rearrangement of gamete chromosomes such as duplication or deletion. Unfortunately, routine techniques such as karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and copy number variation sequencing (CNV-seq) cannot detect all types of SVs. In this study, we show that optical genome mapping (OGM) quickly and accurately detects SVs for RSA patients with a high resolution and provides more information about the breakpoint regions at gene level. Methods: Seven couples who had suffered RSA with unbalanced chromosomal rearrangements of aborted embryos were recruited, and ultra-high molecular weight (UHMW) DNA was isolated from their peripheral blood. The consensus genome map was created by de novo assembly on the Bionano Solve data analysis software. SVs and breakpoints were identified via alignments of the reference genome GRCh38/hg38. The exact breakpoint sequences were verified using either Oxford Nanopore sequencing or Sanger sequencing. Results: Various SVs in the recruited couples were successfully detected by OGM. Also, additional complex chromosomal rearrangement (CCRs) and four cryptic balanced reciprocal translocations (BRTs) were revealed, further refining the underlying genetic causes of RSA. Two of the disrupted genes identified in this study, FOXK2 [46,XY,t(7; 17)(q31.3; q25)] and PLXDC2 [46,XX,t(10; 16)(p12.31; q23.1)], had been previously shown to be associated with male fertility and embryo transit. Conclusion: OGM accurately detects chromosomal SVs, especially cryptic BRTs and CCRs. It is a useful complement to routine human genetic diagnostics, such as karyotyping, and detects cryptic BRTs and CCRs more accurately than routine genetic diagnostics.

OBJECTIVE: Approximately 1% of individuals who carry a balanced reciprocal translocation (BRT) are subfertile. Current karyotyping does not have the resolution to determine whether the breakpoints of the involved chromosomes perturb genes important for fertility. The aim of this study was to apply single-molecule optical mapping (SMOM) to patients presenting for IVF (in vitro fertilization) to ascertain whether the BRT disrupted any genes associated with normal fertility.
METHODS: Nine subfertile patients with different BRTs were recruited for the study. Methyltransferase enzyme DLE1 was used to fluorescently label their genomic DNA samples at the recognition motif CTTAAG. The SMOM was performed on the Bionano platform, and long molecules aligned against the reference genome hg19 to identify the breakpoint regions. Mate-pair and PCR-Sanger sequencing were used to confirm the precise breakpoint sequences.
RESULTS: Both breakpoint regions in each of the nine BRTs were finely mapped to small regions of approximately 10 Kb, and their positions were consistent with original cytogenetic banding patterns determined by karyotyping. In three BRTs, breakpoints disrupted genes known to be associated with male infertility, namely NUP155 and FNDC3A [46,XY,t(5;13)(p15;q22)], DPY19L1 [46,XY,t(1;7)(p36.3;p15), and BAI3 [46,XY,t(3;6)(p21;q16)].
CONCLUSIONS: The SMOM has potential clinical application as a rapid tool to screen patients with BRTs for underlying genetic causes of infertility and other diseases.