Late Onset Alzheimer’s Disease (LOAD) is a common neurodegenerative disorder that affects ~5 million Americans. A primary risk factor in sporadic LOAD is the apolipoprotein E (APOE) gene, with carriers of the É›4 allele (É›3/É›4 heterozygotes or É›4/É›4 homozygotes) being at highest risk compared to É›3/É›3 homozygotes. LOAD is strongly sex-biased, with increased frequency in women (XX), but also increased severity in men (XY). The impact of genetic sex differences combined with APOE genotypes has not been studied. Our goal is to build models with various sex chromosome complements and APOE genotypes to understand interactions of these genetic factors in sex differences in LOAD pathology. The Disteche lab has derived isogenic pairs of human induced pluripotent stem cell (iPSC) lines with different numbers of sex chromosomes, e.g. XY/XXY, X0/XX, or X0/XXX. These pairs are genetically identical, save for their sex chromosomes. My project is to use CRISPR/Cas9 editing to generate different APOE genotypes in these paired lines. Starting from an XXY/XY isogenic pair from a heterozygote É›4/É›3, I am generating É›3/É›3 clones with either the XXY or XY genotype. To accomplish this, I transfect iPSCs with Cas9 and APOE-specific guide RNAs in the presence of a single-stranded DNA donor that contains the É›3 allele. After DNA cleavage and replacement, edited É›3/É›3 single-cell clones and control É›4/É›3 clones are identified using PCR and DNA sequencing, followed by karyotyping and verifying the absence of off-target effects and epigenetic instability. The new isogenic iPSC lines with different APOE genotypes and sex chromosome complements will be differentiated to LOAD-relevant cell types, including neurons, microglia, and cortical organoids, for transcriptomic and functional analyses to better understand how APOE genotypes and genetic sex interact to modulate risk in LOAD pathology.

In mammals, males have sex chromosomes XY while females have XX. To balance out the extra X sex chromosome, females undergo X-chromosome inactivation (XCI) which silences most genes on one of the two X chromosomes. However, some genes escape XCI and continue to be expressed on the inactive X chromosome causing a high expression level of these genes in females compared to XY males, leading to potential sex differences in health and disease. Shroom4, an X-linked gene that encodes an essential protein for cytoskeletal architecture, is an example of a gene that escapes XCI in mice. How Shroom4 escapes XCI is unclear. It has been proposed that CTCF, a master chromatin regulator that controls gene transcription through histone or chromatin modifications, could play a role in insulating escape genes from the silencing environment on the inactive X chromosome. Indeed, we found there is a strong CTCF peak between Shroom4 and the neighbor silenced gene Bmp15. To functionally test this insulation model, I am using CRISPR/Cas9 to edit the CTCF binding site and examining the effects of deletion and inversion of the site on Shroom4 allelic expression levels. This analysis will show whether the CTCF binding site and its correct orientation are necessary for Shroom4 escape from X inactivation. Through this project we are able to improve our understanding of the complex nature of XCI.

Individuals experiencing homelessness and housing instability in King County have inadequate access to water, sanitation, and hygiene (WASH) services. This urgent matter of community health has only been exacerbated by the coronavirus disease (COVID-19) pandemic in recent years and can necessitate open defecation or unsafe disposal of wastewater. Particularly among those residing in recreational vehicles (RVs) and use their bathroom facilities, unsafe sanitation practices increase the risks of intestinal pathogen transmission and infection in densely populated communities. In a continuation of efforts to address this, I have developed a study that aims to identify an optimal method of RV wastewater concentration for downstream detection of pathogens. With informed consent from RV residents and sampling assistance from Seattle Public Utilities, I have began collecting a representative collection of samples of RV wastewater from multiple neighborhoods across Seattle. I have utilized a split-and-seed approach in which half the samples were seeded with known amounts of target bacterial, viral, and spore-forming organisms, and the other half left unseeded as a control. These samples will be used to compare two methods of sample concentration, skimmed milk flocculation and membrane filtration, using a weighted rubric that evaluates biosafety, seeded organism recovery efficiency, personnel time, and cost. Upon observation in the lab, I have found that skimmed milk flocculation led to inhibition during detection via molecular assays. Additionally, high turbidity of the samples yielded significant logistical challenges with processing skimmed milk samples, leading my team and I to instead favor the membrane filtration technique. As we move forward, I anticipate detecting elevated levels of pathogens in wastewater from RVs when compared to samples from control locations chosen to represent populations living in sewered and unsewered environments representing populations. Upon development of the optimized protocol, my work will be prepared for publication in a peer-reviewed journal and shared with the City of Seattle.