May 21, 2021 Mary Gates Hall

GENETIC FOUNDATIONS OF HUMAN DISEASE O-3F Session Moderator: Brandon Berry, Join Room 1:00 PM to 2:30 PM

* Note: Titles in order of presentation.

Is the Inverted Amplification Mechanism of the Yeast observed reduction in rates of SUL1 amplification events with SUL1 Gene Dependent on Proximity to the Telomere? the reconstructed chromosomes would indicate telomeric in- Isabelle Young, Senior, Biology (Molecular, Cellular & fluence on the amplification mechanism prompting further Developmental) examinations within that genomic region. Attaining a greater Louis Stokes Alliance for Minority Participation, understanding of this CNV mechanism yields information for McNair Scholar, UW Honors Program future implications in genetic disease research. Mentor: M.K. Raghuraman, Genome Sciences Mentor: Bonita Brewer, Genome Sciences Identifying New Genes for Congenital Heart Defects Copy number variants (CNVs) are typically a result of chro- Using Protein-Protein Interaction Networks and mosomal duplications and deletions, making them a well- CRISPR Screening known form of genetic diversity and associated with several Whitaker Chamblin Reid, Senior, Biology (Molecular, human disorders. Little is known about CNVs within hu- Cellular & Developmental) mans and insight into CNV mechanisms would help scientists Mentor: Lisa Maves, Pediatrics better understand, and potentially treat, many genome-based diseases. A particular form of CNV within humans is the in- While Congenital Heart Defects (CHDs) are the most com- verted triplication of a gene without any chromosomal dele- mon birth defect in the US, only 20-30% of the genes that tions. A similar phenomenon is observed at the SUL1 gene in contribute to the development of CHDs have been identi- Saccharomyces cerevisiae yeast cells, providing a model for fied. The purpose of this research is to identify the un- studying such CNVs. The Brewer lab proposed a replication known genetic causes of human CHDs through a combina- error mechanism responsible for this specific amplification tion of Protein-Protein Interaction (PPI) network analysis and described as Origin Dependent Inverted Replication Ampli- genetic manipulation of zebrafish using CRISPR. Using the fication (ODIRA). What impacts the initiation of this mech- ExAC human genetic database, our lab has identified over anism is unknown, but the proximity of SUL1 to the telom- 200 new ”Candidate” genes for CHDs. For this project, ere raises the possibility that properties of the telomere may we sought to answer two main questions: (1) Can PPI net- stimulate replication errors responsible for the triplication. I works serve to identify new gene interactions potentially in- conducted a literature review analyzing 11 articles discussing volved in the development of CHDs? (2) Can we demon- various CNV mechanisms and telomeric influence on repli- strate functions and interactions of these new genes using mu- cation to establish their relationship. Through my review, I tant zebrafish embryos? To answer the first question, we uti- found a likely method to test whether the telomere does affect lized STRING—an online database of known and predicted ODIRA. I propose utilizing a CRISPR-Cas9 based method to protein-protein interactions—to create PPI networks that pre- first circularize and eliminate the telomeres of the chromo- dict interactions between our “Candidate” and “Known” some. Subsequently, the chromosome would be linearized at genes involved in human CHDs. Through PPI network anal- a location distant from the original telomere sites, effectively ysis, we identified the interaction of five proteasome factors POMP PSMA6 PSMA7 PSMD3 PSMD6 moving the entire SUL1 site away from potential telomeric ( , , , , and ). The pro- influence. This research design allows for a comparison of teasome system has been characterized to be involved in hu- SUL1 amplification events within the original and the restruc- man cardiac disease, but the specific roles of these factors tured chromosomes and would reveal whether the telomeric in heart development have not yet been determined. To ad- region influences inverted SUL1 amplification formation. An dress our second question, we used CRISPR-Cas9 zebrafish genome editing to preliminarily identify functions of the pro-

Undergraduate Research Program 1 www.uw.edu/undergradresearch teosome factors POMP, PSMA6 and PSMD6 in heart devel- Post-Operative Radiation Therapy to Prevent Local opment. We then used CRISPR to create a new POMP mu- Recurrence of Low-Risk Merkel Cell Carcinomas of the tant zebrafish strain. Our genotypic and phenotypic analyses Head and Neck Versus Other Sites confirm a critical role for POMP in heart development. This Marika Margaret Bierma, Senior, Microbiology, is crucial, as it demonstrates that this cluster of proteasome Comparative History of Ideas factors could potentially be identified as a novel grouping of UW Honors Program genes that are related to human CHDs. Our results promise Mentor: Paul Nghiem, Dermatology to further our understanding of the genetic causes of human Mentor: Peter Goff CHDs. Mentor: Kristina Lachance, Medicine, Dermatology Merkel cell carcinoma (MCC) is a rare, aggressive skin can- Identification and Evolution of the Human ZP3r cer with a recurrence risk of ˜40%; however, prognosis for Ortholog low-risk, stage I disease is excellent with primary surgical Derya Hasena Gurbuz, Freshman, Pre-Health Sciences management. The role of post-operative radiation therapy Mentor: Willie Swanson, Genome Sciences (PORT) is controversial as it can cause significant and acute Mentor: Jolie Carlisle, Genome Sciences long-term side effects. Here, we assess the efficacy of PORT Although fertilization is a crucial process for sexually re- on local recurrence (LR) rates in patients with pathological producing organisms, the molecular mechanisms mediating stage I MCC with primary tumors on the head/neck (HN) vs. this process in humans and other animals remain unknown. non-head and neck (Non-HN) sites. One hundred forty-seven In part, this is due to reproductive proteins evolving rapidly MCC patients treated from 2006-2020 were identified from between closely related species. Sequence diversification an IRB-approved prospective registry who had ‘low-risk’ dis- and gene duplication makes establishing gene orthology (the ease: pathological T1 primary tumor resected with negative same genetic locus in different species) difficult. The step margins, negative pathologic node status, and no immunosup- of fertilization that I study is the sperm binding to the egg pression. LR was defined as tumor recurrence within 2 cm coat. ZP3 is an egg-coat protein and ZP3r is its sperm bind- of the primary surgical bed. I led compilation of the cohort, ing partner in mice. My research has shown that the human and contributed to discussion of results, and development of ortholog of mouse ZP3r has been misidentified as C4BPA and the figures and manuscript. Seventy-nine patients received is instead the pseudogene C4BPAP1. This misidentification PORT (30 HN, 49 Non-HN), and 68 patients were treated is likely due to difficulties in determining orthology when the with surgery alone (30 HN, 38 Non-HN). Addition of PORT gene is surrounded by duplicates. C4BPAP1 shows testes- was associated with a decreased risk of LR across the entire specific expression despite being a pseudogene, consistent cohort (5-year rate: 9.5% vs. 0%, p=0.004), with 6 LRs in with a function in fertilization. However, pseudogenizing mu- the surgery alone group. The addition of PORT significantly tations in the C4BPAP1 locus have accumulated in humans reduced LR rates among HN patients (21% vs. 0%, p=0.034). and other great apes, indicating this gene may no longer play Conversely, no LRs were observed in Non-HN patients. No a role in fertilization in these species. Phylogenetic analysis significant MCC-specific survival differences were observed. of loci performed by maximum likelihood (Phylip) and syn- For low-risk MCC of the extremities and trunk, excellent out- teny analysis shows that C4BPAP1 is the human ortholog of comes were achieved with surgery alone. However, HN MCC mouse ZP3r. By comparing protein sequences across species, was a risk factor for LR that was significantly reduced with we have found that ZP3r/C4BPAP1 varies in the number of PORT. Overall, this study demonstrates the importance of pri- CCP domains. The ancestral form of this gene in mammals mary tumor site location for prognosis and treatment of MCC contains 8 CCP domains, meanwhile mice contain 7. During to determine patients that would benefit from PORT and those my next two quarters, we will examine the evolution of the that can be spared the toxic side effects of radiotherapy. ZP3r gene family, look at ZP3r’s conservation across mam- mals, and determine whether ZP3r is undergoing positive se- lection, a characteristic common to functional fertilization genes. Our research is refining the identification of genetic loci implicated in mammalian fertilization. The precise iden- tification of these loci is important for the development of novel contraceptives and for studies of infertility.

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