Fetal loss by novel ZNHIT3 variants

ZNHIT3 (zinc finger HIT type containing protein 3) is an evolutionarily conserved protein required for ribosome biogenesis by mediating the assembly of small nucleolar RNAs (snoRNAs) of class C/D into ribonucleoprotein complexes (snoRNPs). Missense mutations in the gene encoding ZNHIT3 protein have been previously reported to cause PEHO syndrome, a severe neurodevelopmental disorder typically presenting after birth. We present here the case of two fetuses from a single family who presented with isolated hydrops during the early second trimester of pregnancy, resulting in intrauterine demise. Autopsy revealed no associated malformation. Through whole-genome quartet analysis, we identified two novel variants within the ZNHIT3 gene, both inherited from healthy parents and occurring as compound heterozygotes in both fetuses. The c.40T>C p.Cys14Arg variant originated from the father, while the c.251_254delAAGA variant was of maternal origin. Western capillary analysis of ZNHIT3 protein in lymphoblastoid cell lines of the parents shows that the parental c.40T>C p.Cys14Arg variant causes a significant drop in steady-state ZNHIT3 protein level. Analysis of the variants in human cell culture models reveals that both variants reduce cell growth, albeit to different extents, and impact the protein's stability and function in distinct ways. The c.251_254delAAGA results in production of a stable form of ZNHIT3 that lacks a domain required for mediating snoRNP biogenesis, whereas the c.40T>C p.Cys14Arg variation behaves similarly to the previously described PEHO-associated ZNHIT3 variants that destabilize the protein. Interestingly, both variations lead to a marked decrease in specific box C/D snoRNA levels, reduced rRNA levels and cellular translation. Analysis of rRNA methylation pattern in fetus samples reveals distinct sites of hypo 2'-O-methylation. RNA-seq analysis of undifferentiated and differentiated SHSY5Y cells transfected with the ZNHIT3 variants reveals differential expression of a set of genes, many of which are associated with developmental processes and RNA binding compared to cells expressing wild-type ZNHIT3. In summary, this work adds two novel ZNHIT3 variants to the other human disease-linked variants of this protein and describes the molecular defects caused by these pathogenic variants.