Increased Expression of ZFPM2 Bypasses SRY to Drive 46,XX Testicular Development: A New Mechanism of 46,XX DSD

We present a patient with a novel cause of 46,XX ambiguous/androgenous genitalia Differences of Sex Development (DSD). Genome-wide array from blood showed 46,XX with [~]35% mosaic duplication of 76.5 Mb at chromosome 8q13.2-q24.3, containing 257 OMIM genes including ZFPM2 and CYP11B1. Congenital adrenal hyperplasia testing was negative, testosterone was elevated, and the pro-testicular master regulator SRY was absent. The infant had a uterus, one streak ovary (<5% 8q duplication), and one testicle (75% 8q duplication). We hypothesized that mosaic ZFPM2 duplication resulted in localized ZFPM2 overexpression and testicular development. In typical testicular development, ZFPM2 and its binding partner, GATA4, drive expression of the SRY master regulator. We completed RNA-seq of four Formalin-Fixed Paraffin-Embedded (FFPE) gonadal tissue samples from this mosaic individual to identify differentially expressed genes (DEGs). After quality control, two lines representing with and without the duplication were analyzed. In gonadal tissue containing the duplication, increased dosage of ZFPM2 in a Sry-negative-46-XX individual appears to upregulate transcriptional activity of gonadal specific promoters such as SOX9 and AMH via its protein interaction with known regulator of early testis development GATA4. ZFPM2 is essential to the GATA4/ZFPM2 transcription complex. Our results show that increased ZFPM2 dosage enhances ZFPM2s interaction with GATA4 and results in upregulation of SOX9 that is sufficient to initiate testis differentiation independent of SRY. SOX9 (FC=39.2, p=6.1x10-119) and SF-1 (FC=1.4, p=1.6x10-3) interact to produce the functional marker of fetal Sertoli cells AMH (FC=108.4, p=3.0x10-54) and inhibit female sexual differentiation. Several components of the testicular sex-development pathway were upregulated in addition to SOX9 and AMH, including the pro-testicular transcription factor MAP3K1 (FC=1.7, p=5.6x10-17), DMRT1 (FC=13.9, p=2.1x10-12), LHX9 (FC=2.5, p=5.0x10-14), DHH (FC=12.0, p=1.8x10-30), PTGDS (FC=2.5, p=3.4x10-18), and SOX8 (FC=10.9, p=6.5x10-10). ZFPM2 may function as a master temporal and spatial regulator of mammalian testicular organogenesis whose increased dosage elicits significant and cascading downstream effects. Further, components of the ovarian WNT-signaling pathway were repressed, including LEF1 (FC=-3.7, p=1.4x10-21) and FOXL2 (FC=-8.1, p=2.9x10-39). We have shown that increased ZFPM2 dosage can induce 46,XX testicular development in a manner not dependent on SRY. This contravenes the previous understanding that GATA4/ZFPM2 drives testicular development through SRY. ZFPM2 may modulate numerous critical sex-development genes including transcription factors otherwise thought to be downstream of SRY (MAP3K1, SOX9, AMH). Findings from this single high-yield patient demonstrate that the primary role of ZFPM2 in testicular development may be independent of Sry. This adds ZFPM2 to the brief (<10) list of genes capable of directing testicular development in the 46,XX context, absent SRY. Overall, new understanding of these genes demonstrates that the role of SRY as a "master regulator" of testicular development may be less than previously thought.