Increased Expression ofZFPM2BypassesSRYto Drive 46,XX Testicular Development: A New Mechanism of 46,XX DSD
Authors: Ragno Leah , Yang Daphne , Bhatti Trisha R. , Bradfield Jonathan P. , Bowen Diane K. , Grand Katheryn L. , Hakonarson Hakon , Kolon Thomas F. , Glessner Joseph , Simon M. Celeste , Skuli Nicolas , Vogiatzi Maria G. , Guerraty Marie A. , Deardorff Matthew A. , Pyle Louise C. 
Published: May 2024

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 includingZFPM2andCYP11B1. Congenital adrenal hyperplasia testing was negative, testosterone was elevated, and the pro-testicular master regulatorSRYwas 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,ZFPM2and its binding partner,GATA4, drive expression of theSRYmaster 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 ofZFPM2in aSry-negative-46-XX individual appears to upregulate transcriptional activity of gonadal specific promoters such asSOX9andAMHvia its protein interaction with known regulator of early testis developmentGATA4. ZFPM2is essential to theGATA4/ZFPM2transcription complex. Our results show that increasedZFPM2dosage enhancesZFPM2’s interaction withGATA4and results in upregulation ofSOX9that is sufficient to initiate testis differentiation independent ofSRY. SOX9(FC=39.2,p=6.1×10−119) andSF-1(FC=1.4,p=1.6×10−3) interact to produce the functional marker of fetal Sertoli cellsAMH(FC=108.4,p=3.0×10−54) and inhibit female sexual differentiation. Several components of the testicular sex-development pathway were upregulated in addition toSOX9andAMH, including the pro-testicular transcription factorMAP3K1(FC=1.7,p=5.6×10−17),DMRT1(FC=13.9,p=2.1×10−12),LHX9(FC=2.5,p=5.0×10−14),DHH(FC=12.0,p=1.8×10−30),PTGDS(FC=2.5,p=3.4×10−18), andSOX8(FC=10.9,p=6.5×10−10).ZFPM2may 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 ovarianWNT-signaling pathway were repressed, includingLEF1(FC=-3.7,p=1.4×10−21) andFOXL2(FC=-8.1,p=2.9×10−39). We have shown that increasedZFPM2dosage can induce 46,XX testicular development in a manner not dependent onSRY. This contravenes the previous understanding thatGATA4/ZFPM2drives testicular development throughSRY. ZFPM2may modulate numerous critical sex-development genes including transcription factors otherwise thought to be downstream ofSRY(MAP3K1, SOX9, AMH). Findings from this single high-yield patient demonstrate that the primary role ofZFPM2in testicular development may be independent ofSry. This addsZFPM2to the brief (<10) list of genes capable of directing testicular development in the 46,XX context, absentSRY. Overall, new understanding of these genes demonstrates that the role ofSRYas a “master regulator” of testicular development may be less than previously thought.