FRI387 Reproductive Capacity Of Znhit3 Knockout Mouse Model
Authors: Kira Dina , Hawkins Zoe A , Chorich Lynn Parson , Brakta Soumia , Gan Lin , Layman Lawrence Clarke 
Published: October 2023

Disclosure: D. Kira: None. Z.A. Hawkins: None. L.P. Chorich: None. S. Brakta: None. L. Gan: None. L.C. Layman: None.

Introduction: Zinc finger, HIT domain containing 3 (ZNHIT3), also known as thyroid hormone receptor-interacting protein 3 (TRIP-3), is a 155 amino acid protein with an N-terminus Zinc finger HIT domain (11-42a.a) that is involved in pre-ribosomal RNA processing. It also has a C-terminus short conserved LxxLL binding motif that interacts with the ligand binding domain of the thyroid receptor in the presence of thyroid hormone as a co-activator associated with hepatocyte nuclear factor-4α (HNF-4α). ZNHIT3 is localized to the cytoplasm and the nucleus. Biallelic ZNHIT3 pathogenic variants in the N-terminus have been identified in persons with Progressive encephalopathy with Edema, Hypsarrhythmia, and Optic atrophy (PEHO) syndrome, a severe autosomal recessive neurodevelopmental disorder characterized by extreme cerebellar atrophy due to almost total loss of granule neurons. Heterozygous pathogenic variants in ZNHIT3 affecting the C-terminus have been demonstrated in individuals with Mayer-Rokitansky-Küster-Hauser (MRKH). MRKH consists of the congenital absence of the uterus and vagina, either as isolated Mullerian aplasia (type 1) or associated with renal, skeletal, cardiac, and auditory anomalies (type 2). Previous studies of animal models utilizing morpholino injection-induced knockdown and CRISPR editing of znhit3 in zebrafish both showed cerebellar defects and microcephaly. We are not aware of a mouse model of Znhit3.Hypothesis: We hypothesize that the Znhit3 knockout (KO) mouse will demonstrate reproductive abnormalities and an MRKH-related phenotype. Methods: Heterozygous Znhit3 (Znhit3+/-) KO mice were generated by CRISPR. A 1 base deletion was introduced in exon 2, creating a frameshift and a stop codon in exon 3, which leads to a truncated protein. We then crossed male and female Znhit3+/- mice and Znhit3+/- and wild-type Znhit3+/+ (WT) mice. Pups' genotypes were confirmed with DNA sequencing of tail samples. Pups were weighed weekly from age 3 weeks to 16 weeks. Results: Mating 17 pairs of Znhit3+/- mice resulted in a total of 73 pups born, of which 50 were Znhit3+/- and, 23 were WT with a ratio of almost 2:1. No homozygous Znhit3 (Znhit3-/-) KO pups were born. There was no significant difference in weights between Znhit3+/- and WT pups. Conclusion: Heterozygous Znhit3+/- animals appeared to be phenotypically normal with similar weights to WT. However, the Hardy-Weinberg equilibrium (1 homozygous wild-type: 2 heterozygous: 1 homozygous variant) was disrupted, as no Znhit3−/− homozygotes were born, suggesting embryonic lethality. It is possible that central nervous system, cardiac, and/or renal defects could be responsible for this lethality, which will be studied in embryos at E18.5, 15.5, and 12.5. A detailed phenotypic characterization will be performed with regard to reproductive function and MRKH-related organ development.

Presentation: Friday, June 16, 2023