ZNF423 patient variants, truncations, and in-frame deletions in mice define an allele-dependent range of midline brain abnormalities

Abstract

Interpreting rare variants remains a challenge in personal genomics, especially for disorders with several causal genes and for genes that cause multiple disorders. ZNF423 encodes a transcriptional regulatory protein that intersects several developmental pathways. ZNF423 has been implicated in rare neurodevelopmental disorders, consistent with midline brain defects in Zfp423-mutant mice, but pathogenic potential of most patient variants remains uncertain. We engineered~50 patient-derived and small deletion variants into the highlyconserved mouse ortholog and examined neuroanatomical measures for 791 littermate pairs. Three substitutions previously asserted pathogenic appeared benign, while a fourth was effectively null. Heterozygous premature termination codon (PTC) variants showed mild haploabnormality, consistent with loss-of-function intolerance inferred from human population data. In-frame deletions of specific zinc fingers showed mild to moderate abnormalities, as did low-expression variants. These results affirm the need for functional validation of rare variants in biological context and demonstrate cost-effective modeling of neuroanatomical abnormalities in mice.

Zinc Finger Proteins in Neuro-Related Diseases Progression

“…Studies have shown that MSUT2 knockout has anti-inflammatory and neuroprotective effects in a mouse model. The flowchart is Frankel et al, 1987;Wolfe et al, 2000;Schefe et al, 2006;Brayer et al, 2008;Town et al, 2009;Fan et al, 2012;Shi et al, 2013;Yuan et al, 2013;Xu et al, 2016;Yu et al, 2017;Brahmachari et al, 2019;Kruszka et al, 2019;Lorsch et al, 2019;Ohkubo et al, 2019;Zhang et al, 2019;Choi et al, 2020;Deshpande et al, 2020;Hu et al, 2020;Xiao et al, 2020;Dentici et shown in Figure 1. Loss of function of MSUT2 can reduce the formation of tau protein and protect the loss of neurons, but the molecular mechanism is still unclear.…”

Smoothened and ARL13B are critical in mouse for superior cerebellar peduncle targeting

“…In addition to defects in the SCPs, the MTS is due to an underdeveloped cerebellar vermis, so we examined the size of the cerebellum and the cerebellar vermis (Figure 5) (Aguilar et al 2012). To quantify cerebellar size, we performed analysis of surface-facing anatomical measurements validated to be sufficiently sensitive to detect small differences in cerebellar vermis size (Deshpande et al 2020). Briefly, we measured cerebellar width as well as cerebellar vermis width (widest part of lobule VII) of fixed whole mount dissected brains.…”

“…Weanling age (P20-P24) male and female mice were sacrificed and brains were harvested and fixed in 4% PFA overnight at 4⁰. Ten sex-matched pairs of mouse brains were collected for each genotype, which is sufficiently powered to detect differences as small as 3% (Deshpande et al 2020). Brains were imaged on a tilted stage to present a surface view of the cerebellum, with a standard ruler in frame to confirm scale, using a dissecting microscope (Leica MZFLIII).…”

“…Whole cerebellar width was measured at the widest part of the cerebellum, coinciding with lobule CI or CII. Vermis width was calculated by measuring the widest part of lobule VII (Deshpande et al 2020). For each sex- and age-matched pair, the ratio of mutant to control width was calculated and a one-sample t-test was performed and compared to a hypothetical value of 1 (PRISM 8.2.0).…”

“…Loss of either of the JSRD-linked genes Ahi1 or Cep290 in mouse leads to a small cerebellar vermis due to aberrant Wnt signaling (Lancaster et al 2011; Ramsbottom et al 2020). JSRD-causing mutations in Znf423 are linked to defects in Wnt, BMP and retinoic acid signaling (Hata et al 2000, Huang et al 2009, Casoni et al 2020, Deshpande et al 2020). Conditional Arl13b or Inpp5e deletion in the SCPs results in their disorganization and thickening through misregulation of ciliary PI3 kinase and AKT (Guo et al 2019).…”