Development of a Clinically Applicable High-Resolution Assay for Sperm Mosaicism

Sperm mosaicism, the presence of a pathogenic variant in a subset of sperm, is an important cause of heritable genetic disease. However, clinical testing for sperm mosaicism outside research has been limited by the lack of Clinical Laboratory Improvement Amendments (CLIA)-validated results deliverable to patients. We developed the Sensitive Assay for Mosaicism (SAM), a two-phase method for sperm mosaicism detection. In phase 1, sperm DNA undergoes deep sequencing using next-generation sequencing or nanopore-based sequencing with unique molecular identifiers (UMIs) to improve accuracy. In phase 2, PCR primers specific to UMI sequences generate amplicons for CLIA-validated Sanger sequencing, providing patient-ready results. SAM's performance was characterized and tested on semen samples from 14 participants, each with a prior offspring with a de novo pathogenic variant. SAM demonstrated a detection limit of approximately 0.005%. The UMI strategy improved sequencing accuracy on next-generation sequencing and nanopore platforms from 99.9% to >99.999%, and from 93% to >99.99%, respectively. Sperm mosaicism was identified in two tested cases: FAM111A (5.51%) and FGFR3 (0.0129%), with FGFR3 exhibiting selfish mutation validated in unrelated individuals showing varying mosaicism levels. SAM provides sensitive detection of low-level sperm mosaicism with CLIA-validated results for patients, enabling recurrence risk assessment and guiding risk mitigation strategies such as in vitro fertilization with preimplantation genetic testing for monogenic disease, sperm donation, and prenatal diagnosis.