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

Significance StatementThis study identifies microhomology-mediated end joining (MMEJ) as a novel mutational mechanism underlying a pathogenic deletion in the androgen receptor gene in a patient with complete androgen insensitivity syndrome. By mapping the genomic breakpoint at the nucleotide level, we demonstrate the presence of canonical features of MMEJ, thereby expanding the known mechanisms of genomic structural variation in AR. These findings underscore the importance of incorporating copy number variation (CNV) detection and breakpoint analysis into diagnostic workflows for 46,XY differences of sex development (DSD), enabling more accurate molecular classification and improved patient management. BackgroundCopy 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. ObjectiveTo 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. MethodsWhole-exome sequencing, Multiplex Ligation-dependent Probe Amplification, PCR, and Sanger sequencing were used to identify and refine a hemizygous deletion affecting exons 6-8 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-specific CNV burden. ResultsAn accurate genomic analysis revealed an 8-bp microhomology region flanking the genomic breakpoint of the CNV event found in this CAIS patient, consistent with microhomology-mediated end joining event. Among 991 AIS cases, 49 harbored AR CNVs, significantly enriched compared to controls (OR = 4.59, P = 9.2 x 10-{superscript 1}). Exon 2 was the most frequently affected region and most strongly associated with CAIS. ConclusionThis 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.