SUN-039 - Characterization of A PEHO-Associated ZNHIT3 Biallelic Variant in Mouse Cerebellar and Reproductive Development

Background:

It is well known that insults of the central nervous system may result or accompany reproductive disorders such as hypogonadotropic hypogonadism and primary ovarian insufficiency, among others. Pathogenic biallelic variants in the amino-terminus of ZNHIT3 are an established cause of progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO), a neurodevelopmental disorder characterized by early and severe cerebellar involvement, whereas heterozygous variants in the carboxy-terminus have been identified in mullerian aplasia in Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. We hypothesize that a mouse model of the PEHO missense variant will demonstrate cerebellar pathology, but the reproductive function is unknown.

Methods:

A knock-in mouse model carrying the human PEHO-associated Znhit3 c.92C>T (p.Ser31Leu) variant was generated by CRISPR to characterize neurodevelopmental and reproductive consequences of Znhit3 disruption. Cerebellar tissue from homozygous (HMZ) mutant and wild-type (WT) mice was examined using hematoxylin and eosin staining to assess Purkinje cell (PC) organization and granule cell density. PC density was calculated as the total number of PCs divided by the length of the PC layer. Reproductive function was evaluated through controlled breeding experiments assessing fertility, litter size, and perinatal survival.


Results:

HMZ Znhit3 mutant female mice exhibited a significant reduction in PC density within the Simple lobule. PC yielded a mean density of 36.14 ± 2.08 PCs/mm in WT mice (n = 5) versus 30.95 ± 2.09. PCs/mm in Znhit3−/− mice (n = 6; p = 0.0345), representing a mean difference of −5.19 ± 2.09 PCs/mm (95% CI −9.91 to −0.47). This reduction is consistent with PEHO-associated cerebellar pathology.

HMZ females remained fertile with a mean litter size of 5.84 ± 2.23 (median 6; IQR 4–7) across more than 50 litters, as well as increased perinatal loss relative to controls. In comparison, WT females typically produced 7–8 pups per litter (n = 50 litters). Reproductive outcomes were consistent across multiple breeding cycles, indicating a reproducible effect of the Znhit3 variant on perinatal viability. Ongoing analyses are assessing genotype-specific survival patterns.

Conclusion:

We describe an in vivo model of a PEHO Znhit3 variant that is associated with disrupted cerebellar development, but reproductive outcomes remain normal. This model provides a foundation for future studies examining the contribution of ZNHIT3 to coordinated neuroendocrine and reproductive development.

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