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

Area of Science:

• Reproductive Genetics
• Molecular Biology
• Clinical Diagnostics

Background:

• Sperm mosaicism, where pathogenic variants occur in a subset of sperm, is a significant cause of inherited genetic disorders.
• Clinical diagnosis of sperm mosaicism has been hindered by the absence of validated, patient-reportable results.

Purpose of the Study:

• To develop and validate a sensitive, CLIA-validated assay for detecting low-level sperm mosaicism.
• To enable accurate recurrence risk assessment for couples experiencing de novo pathogenic variants.

Main Methods:

• Developed the Sensitive Assay for Mosaicism (SAM), a two-phase method involving deep sequencing with unique molecular identifiers (UMIs) and CLIA-validated Sanger sequencing.
• Utilized next-generation sequencing and nanopore sequencing platforms with UMIs to enhance accuracy.
• Tested SAM on semen samples from 14 participants with prior offspring affected by de novo pathogenic variants.

Main Results:

• SAM demonstrated a detection limit of approximately 0.005%, significantly improving sequencing accuracy.
• Unique molecular identifiers (UMIs) increased accuracy on NGS from 99.9% to >99.999% and on nanopore from 93% to >99.99%.
• Sperm mosaicism was identified in two cases (FAM111A at 5.51% and FGFR3 at 0.0129%), with FGFR3 validating as a selfish mutation.

Conclusions:

• The Sensitive Assay for Mosaicism (SAM) offers sensitive and accurate detection of low-level sperm mosaicism with CLIA-validated results.
• SAM facilitates genetic counseling, recurrence risk assessment, and informed reproductive decisions, including IVF with PGT-M, sperm donation, and prenatal diagnosis.