Microhomology-Mediated End Joining as a Novel Mechanism Underlying Androgen Insensitivity Syndrome

Background: Copy number variations in the androgen receptor gene are an underrecognized cause of androgen insensitivity syndrome. Understanding their mutational mechanisms can improve AIS diagnosis and genotype phenotype correlation. Objective: To investigate microhomology mediated end joining (MMEJ) as a mutational mechanism underlying a structural variant in the AR gene and to assess the contribution of AR CNVs to AIS through comparative gene burden analysis. Methods: Whole exome sequencing, Multiplex Ligation dependent Probe Amplification, PCR, and Sanger sequencing were used to identify and refine a hemizygous deletion affecting exons 6, 7, and 8 of the AR gene in a 46,XY individual with CAIS. Breakpoint mapping and local alignment were performed using R packages Biostrings and GenomicRanges. A literature and database review identified AR CNVs in AIS cases, which were compared to CNVs in the general population to assess AR CNV burden. Results: An accurate genomic analysis revealed an 8bp microhomology region flanking the genomic breakpoint of the CNV event found in this CAIS patient, consistent with a microhomology-mediated end-joining event. Among 991 AIS cases, 49 harbored AR CNVs, significantly enriched compared to controls (OR 4.59). Exon 2 was the most frequently affected region and showed the strongest association with CAIS. Conclusion: This study provides the first molecular evidence that MMEJ can mediate pathogenic deletions in the AR gene. The significant enrichment of CNVs in AIS and their non random distribution across functional domains support their role in disease pathogenesis and highlight the value of CNV level analysis in the diagnostic evaluation of AIS.