Copy number variants (CNVs) are typically a result of chromosomal duplications and deletions, making them a well-known form of genetic diversity and associated with several human disorders. Little is known about CNVs within humans and insight into CNV mechanisms would help scientists better understand, and potentially treat, many genome-based diseases. A particular form of CNV within humans is the inverted triplication of a gene without any chromosomal deletions. A similar phenomenon is observed at the SUL1 gene in Saccharomyces cerevisiae yeast cells, providing a model for studying such CNVs. The Brewer lab proposed a replication error mechanism responsible for this specific amplification described as Origin Dependent Inverted Replication Amplification (ODIRA). What impacts the initiation of this mechanism is unknown, but the proximity of SUL1 to the telomere raises the possibility that properties of the telomere may stimulate replication errors responsible for the triplication. I conducted a literature review analyzing 11 articles discussing various CNV mechanisms and telomeric influence on replication to establish their relationship. Through my review, I found a likely method to test whether the telomere does affect ODIRA. I propose utilizing a CRISPR-Cas9 based method to first circularize and eliminate the telomeres of the chromosome. Subsequently, the chromosome would be linearized at a location distant from the original telomere sites, effectively moving the entire SUL1 site away from potential telomeric influence. This research design allows for a comparison of SUL1 amplification events within the original and the restructured chromosomes and would reveal whether the telomeric region influences inverted SUL1 amplification formation. An observed reduction in rates of SUL1 amplification events with the reconstructed chromosomes would indicate telomeric influence on the amplification mechanism prompting further examinations within that genomic region. Attaining a greater understanding of this CNV mechanism yields information for future implications in genetic disease research.

While Congenital Heart Defects (CHDs) are the most common birth defect in the US, only 20-30% of the genes that contribute to the development of CHDs have been identified. The purpose of this research is to identify the unknown genetic causes of human CHDs through a combination of Protein-Protein Interaction (PPI) network analysis and genetic manipulation of zebrafish using CRISPR. Using the ExAC human genetic database, our lab has identified over 200 new "Candidate" genes for CHDs. For this project, we sought to answer two main questions: (1) Can PPI networks serve to identify new gene interactions potentially involved in the development of CHDs? (2) Can we demonstrate functions and interactions of these new genes using mutant zebrafish embryos? To answer the first question, we utilized STRING—an online database of known and predicted protein-protein interactions—to create PPI networks that predict interactions between our “Candidate” and “Known” genes involved in human CHDs. Through PPI network analysis, we identified the interaction of five proteasome factors (POMP, PSMA6, PSMA7, PSMD3, and PSMD6). The proteasome system has been characterized to be involved in human cardiac disease, but the specific roles of these factors in heart development have not yet been determined. To address our second question, we used CRISPR-Cas9 zebrafish genome editing to preliminarily identify functions of the proteosome factors POMP, PSMA6 and PSMD6 in heart development. We then used CRISPR to create a new POMP mutant zebrafish strain. Our genotypic and phenotypic analyses confirm a critical role for POMP in heart development. This is crucial, as it demonstrates that this cluster of proteasome factors could potentially be identified as a novel grouping of genes that are related to human CHDs. Our results promise to further our understanding of the genetic causes of human CHDs.

Although fertilization is a crucial process for sexually reproducing organisms, the molecular mechanisms mediating this process in humans and other animals remain unknown. In part, this is due to reproductive proteins evolving rapidly between closely related species. Sequence diversification and gene duplication makes establishing gene orthology (the same genetic locus in different species) difficult. The step of fertilization that I study is the sperm binding to the egg coat. ZP3 is an egg-coat protein and ZP3r is its sperm binding partner in mice. My research has shown that the human ortholog of mouse ZP3r has been misidentified as C4BPA and is instead the pseudogene C4BPAP1. This misidentification is likely due to difficulties in determining orthology when the gene is surrounded by duplicates. C4BPAP1 shows testes-specific expression despite being a pseudogene, consistent with a function in fertilization. However, pseudogenizing mutations in the C4BPAP1 locus have accumulated in humans and other great apes, indicating this gene may no longer play a role in fertilization in these species. Phylogenetic analysis of loci performed by maximum likelihood (Phylip) and synteny analysis shows that C4BPAP1 is the human ortholog of mouse ZP3r. By comparing protein sequences across species, we have found that ZP3r/C4BPAP1 varies in the number of CCP domains. The ancestral form of this gene in mammals contains 8 CCP domains, meanwhile mice contain 7. During my next two quarters, we will examine the evolution of the ZP3r gene family, look at ZP3r’s conservation across mammals, and determine whether ZP3r is undergoing positive selection, a characteristic common to functional fertilization genes. Our research is refining the identification of genetic loci implicated in mammalian fertilization. The precise identification of these loci is important for the development of novel contraceptives and for studies of infertility.
 

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with a recurrence risk of ~40%; however, prognosis for low-risk, stage I disease is excellent with primary surgical management. The role of post-operative radiation therapy (PORT) is controversial as it can cause significant and acute long-term side effects. Here, we assess the efficacy of PORT on local recurrence (LR) rates in patients with pathological stage I MCC with primary tumors on the head/neck (HN) vs. non-head and neck (Non-HN) sites. One hundred forty-seven MCC patients treated from 2006-2020 were identified from an IRB-approved prospective registry who had ‘low-risk’ disease: pathological T1 primary tumor resected with negative margins, negative pathologic node status, and no immunosuppression. LR was defined as tumor recurrence within 2 cm of the primary surgical bed. I led compilation of the cohort, and contributed to discussion of results, and development of the figures and manuscript. Seventy-nine patients received PORT (30 HN, 49 Non-HN), and 68 patients were treated with surgery alone (30 HN, 38 Non-HN). Addition of PORT was associated with a decreased risk of LR across the entire cohort (5-year rate: 9.5% vs. 0%, p=0.004), with 6 LRs in the surgery alone group. The addition of PORT significantly reduced LR rates among HN patients (21% vs. 0%, p=0.034). Conversely, no LRs were observed in Non-HN patients. No significant MCC-specific survival differences were observed. For low-risk MCC of the extremities and trunk, excellent outcomes were achieved with surgery alone. However, HN MCC was a risk factor for LR that was significantly reduced with PORT. Overall, this study demonstrates the importance of primary tumor site location for prognosis and treatment of MCC to determine patients that would benefit from PORT and those that can be spared the toxic side effects of radiotherapy.