Investigating the role of eef1a2 in zebrafish as a potential disease model

Eukaryotic elongation factor (eEF1A) plays a vital role in protein synthesis. It recruits amino-acylated tRNAs and delivers them to the ribosome during protein translation. eEF1A is conserved throughout evolution and exists as independently encoded isoforms in many species. In mammals, there are two isoforms: eEF1A1 and eEF1A2. Unlike eEF1A1 which is widely expressed, expression of eEF1A2 is restricted to the brain, heart and skeletal muscle and is upregulated during development. In mice, homozygote deletion in Eef1a2 gene resulting in the complete loss of function of eEF1A2 causes severe neurodegeneration, loss of muscle bulk and death by 28 days. Recently, de novo heterozygous missense mutation in EEF1A2 has been identified in humans which cause epilepsy, autism and intellectual disability. The main aim of this project was to investigate the use of zebrafish as a model to better understand the role of eEF1A2 in neurological disorders. In addition to its many advantages, the zebrafish has been shown to be an excellent tool for in vivo drug screening. This is an attractive attribute for our studies as regards developing treatment strategies for these disorders. Zebrafish possess four eef1a genes: eef1a1l1, eef1a1a, eef1a1b and eef1a2 which encodes separate highly similar proteins: eEF1A1L1, eEF1A1A, eEF1A1B and eEF1A2 respectively. The zebrafish eEF1A2 shares a 94% sequence identity with the mouse and human eEF1A2 at the amino acid level. In this work, characterisation of zebrafish eEF1A genes was first carried out, as there is currently little information available. Using conventional reverse transcriptase polymerase chain reaction (RTPCR) and real time quantitative PCR (qPCR), I analysed the expression pattern of eef1a genes at different embryonic stages and adult tissues. These genes were differentially expressed with only eef1a1l1 detected at earlier developmental stages, followed by eef1a1a and eef1a1b. Similar to mammals, eef1a2 is detected much later (48 hpf) during development. Co-expression of eef1a mRNA was observed in the adult tissues analysed except in liver where eef1a2 was not detected. An attempt to knock-in one of the epilepsy causing variant, G70S into the zebrafish genome using CRISPR/Cas9 technology was unsuccessful. However, I established two null eef1a2 mutant lines using this technology. Homozygotes from these null lines showed no obvious phenotype and in contrast to null Eef1a2 mice, they are fertile and viable through adulthood. No evidence of neurodegeneration was observed. These results suggest the possibility of compensatory mechanisms activated by the other eef1a genes to buffer the loss of eef1a2 in the mutants. However, preliminary findings suggest that eef1a null mutation might cause zebrafish to be susceptible to PTZ-induced seizures. Results from this work has provided vital information on functional redundancy of eef1a genes in zebrafish and a foundation for further validation of the zebrafish as a model system

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.−339_361dup for CHED1 and c.−370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.−274T>G and c.−307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies