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Abstract

Abstract:

This Abstract contains text that is based closely on, or identical to, text found in my Junior Paper [Steidl, W. “Investigation of the Effects of ppp1cb Clinical Mutations on Zebrafish Embryo Phenotype and Regulation of the Ras/MAPK Signaling Pathway,” Junior Independent Work Paper, Spring 2019].

RASopathies are developmental disorders caused by genetic missense mutations in genes of the RAS/MAPK signaling pathway. Patients with these syndromes often display characteristic symptoms like dysmorphic features, congenital heart abnormalities, and intellectual impairment. Research has shown that the protein phosphatase-1 catalytic subunit beta (PPP1CB) gene could be important to understanding how RASopathies arise. This gene encodes for a subunit of protein phosphatase-1 (PP1), which dephosphorylates elements of the Ras/MAPK pathway. Genetic missense mutations in PPP1CB have been shown to cause a RASopathy called Noonan Syndrome with Loose Anagen Hair. This occurs mechanistically through the enhancement of PP1-SHOC2-MRAS complex formation, producing upregulation of RAF and thus enhancement of the Ras/MAPK pathway. However, there are several other clinically relevant PPP1CB mutations whose mechanisms have not yet been defined. In this paper, I sought to examine the phenotypic effects of clinically relevant mutations in zebrafish and compare them to clinical symptoms using two strategies. First, I aimed to assess the phenotypic effects observed at overexpressed gene levels through injection of embryos with PPP1CB mRNA containing clinical mutations. Second, I aimed to observe phenotypic effects at endogenous expression levels by using CRISPR technology to both knock out the gene and to introduce clinical mutations into ppp1cb. Though my research was cut short due to the coronavirus pandemic, I found that F0 CRISPR-knockout zebrafish mosaic mutants show phenotypic evidence of shortened body length, craniofacial abnormalities, cardiovascular edema. In addition, both F2 CRISPR knockout and knock-in zebrafish homozygous mutants show phenotypic evidence of irregular tail bending and cardiovascular edema, with knockouts also showing evidence of shortened body length. These phenotypes indicate a strong likelihood that ppp1cb has been mutated in zebrafish and demonstrate that ppp1cb mutations lead to RASopathy-like symptoms.