Biology subjects

Lara, R. Z.

Publications and source records attributed to Lara, R. Z..

2 recordsLinked to original sources

bioRxiv2025

Tulp3 quantitative alleles titrate requirements for viability, brain development, and kidney homeostasis but do not suppress Zfp423 mutations in mice.

Tubby-like protein 3 (TULP3) regulates receptor trafficking in primary cilia and antagonizes SHH signaling. Tulp3 knockout mice are embryonic lethal with developmental abnormalities in multiple organs, while tissue-specific knockouts and viable missense alleles cause polycystic kidney disease. Human patients with TULP3 mutations present with variable, but often multi-organ fibrotic disease. We previously showed that mouse and human Tulp3 expression is negatively regulated by ZNF423, which is required for SHH sensitivity in some progenitor cell models. The level of TULP3 function required to prevent mutant phenotypes has not been known. Here we report a Tulp3 quantitative allelic series, designed by targeting the polypyrimidine tract 5 to the splice acceptor of a critical exon, that shows distinct dose-response effects on viability, brain overgrowth, weight gain, and cystic kidney disease. We find limited evidence for genetic interaction with Zfp423 null or hypomorphic mutations. Together, these results establish an approach to developing quantitative allelic series by exon exclusion, rank-order dose-sensitivity of Tulp3 phenotypes, and model thresholds for TULP3 function to prevent severe outcomes. Author SummaryTULP3 protein plays critical roles in regulating receptor trafficking and signaling in the primary cilium. Mutations in the TULP3 gene can cause severe, multi-organ disorders in both mice and humans, yet the amount of TULP3 activity needed to avoid these outcomes has been unclear. In this study, we used precise genome editing in mice to create a set of new Tulp3 gene variants that reduce TULP3 expression to varying degrees. This allowed us to test how much TULP3 is required for survival, normal brain and kidney development, and weight regulation. We found that as little as 5% of normal TULP3 levels is enough to avoid lethal birth defects, but still leads to obesity, mild brain overgrowth, and progressive kidney cysts preceded by reductions in cilium frequency and length in situ. The severity of these effects was related to TULP3 protein levels, highlighting a dose-dependent response. We also investigated whether reducing TULP3 levels would suppress brain abnormalities in Zfp423 mutant mice, based on prior evidence of a genetic interaction, but did not find evidence to support this effect. Our work provides a framework for understanding how varying levels of TULP3 affect various organ systems and offers a general strategy for creating quantitative genetic models of human disease.

genetics↗

bioRxiv2020

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

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 highly-conserved 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. AUTHOR SUMMARYGene identification in rare disorders is typically supported by finding different mutations of the same gene in multiple families with the same disorder. However, causal evidence for any specific mutation found in one or a few related individuals is weaker, especially if the disorder can be caused by any of several genes and the functional effect of the mutation is not certain. Experimental models can be helpful in testing causal effects, but only to the extent that the model is validated to recapitulate one or more aspects of the disorder. We used CRISPR/Cas9-based genome engineering to create a wide range of mutations in mouse Zfp423, whose human cognate is implicated in neurodevelopmental disorders, especially cerebellar vermis hypoplasia and Joubert syndrome. This large collection of animal models shows that both reduced Zfp423 expression, including heterozygosity for loss-of-function mutations, and normally-expressed domain deletions, including specific zinc finger domains, produce measureable abnormalities in midline development. Despite this high level of validation, most patient-derived amino acid substitution variants tested do not produce measureable effects. The single exception is a substitution, H1277Y, that destroys a structural element in the last zinc finger domain and results in dramatic loss of steady-state Zfp423 protein level.

genetics↗