Breakpoint Mapping and Disrupted Gene Identification in Azoospermic Patients With Chromosomal Balanced Translocation by Nanopore Sequencing

OBJECTIVE: To evaluate the utility of nanopore long-read sequencing for precise breakpoint mapping in azoospermia patients with chromosomal balanced translocations, and to elucidate the molecular mechanisms underlying translocation-induced spermatogenic failure. METHODS: Peripheral blood specimens were collected from two azoospermic patients with balanced chromosomal translocations. G-banding karyotype analysis and chromosomal microarray analysis (CMA) results served as the reference standard, and whole-genome long-read sequencing of peripheral blood samples was performed on the PromethION platform. Breakpoint positions were visualized using the Integrative Genomics Viewer (IGV), and gene annotations were conducted using the UCSC Genome Browser. RESULTS: Sequencing was successfully completed for both samples, yielding mean read lengths of 9713.3 bp and 8129.8 bp, respectively. Nanopore sequencing afforded superior molecular resolution of chromosomal aberrations relative to traditional cytogenetic approaches. A total of eight breakpoints were identified through IGV visualization, with four precisely mapped at single-nucleotide resolution. Annotation revealed that five breakpoints resided within intergenic regions without compromising gene integrity, whereas three breakpoints (ChrX:147,998,514-147,998,515, Chr3:65,894,036-65,899,651, and Chr16:53,815,988-53,815,992) were situated within intronic segments of the AFF2, MAGI1, and FTO genes, respectively, resulting in gene disruption. CONCLUSION: This study employed nanopore long-read sequencing to precisely map chromosomal breakpoints in two azoospermic patients, including cryptic breakpoints undetectable by conventional karyotyping. Disruption of AFF2, MAGI1, and FTO at three breakpoints provides novel candidate gene evidence for translocation-associated spermatogenic failure, confirming nanopore sequencing as a robust tool for structural variant characterization and the discovery of spermatogenesis-related genes.