Charcot-Marie-Tooth (CMT) is a group of inherited nerve disorders resulting in muscle weakness in which the motor and/or sensory peripheral nerves are affected; hereditary neuralgic amyotrophy (HNA) is a rare genetic disorder characterized by severe pain and muscle wasting in the arms. Using whole exome sequencing (WES), we aimed to find the causative mutation in a five-generation family with CMT and single-generation family with HNA. WES is an efficient method to identify possible disease-causing mutations by searching for variations in the protein-coding region of any gene. After excluding variants from a known list of genes to be causative of CMT, we used stepwise criteria to filter and prioritize the candidate variants in the exome data from two patients in this family: heterozygosity, alternative splicing, gnomAD frequency, CADD score, function of the gene, and its expression. In CMT, out of the 26 candidate variants, 10 were INDEL mutations and 16 were single nucleotide polymorphisms (SNPs). Each variant was Sanger sequenced to verify the exome variant; the verified variants were further evaluated for co-segregation with the disease by Sanger sequencing affected and unaffected individuals of the family. The results from these analyses did not identify any candidate variant as a possible candidate, concluding the CMT WES study without yielding a causative gene. Our next steps for this project will be to use whole genomic sequencing (WGS) to reveal DNA variation outside the exome or copy number variant or repeat expansion that may alter protein production to identify potential causative mutations. Currently, we are using the same methodology to screen for candidate variants in three exomes from the family with HNA. Out of 22 candidate variants, we have screened 2 INDEL mutations and 2 SNPs. We are actively screening the remaining variants in search of a causative mutation for HNA in this family.

Cancer is an evolutionary process driven by mutagenesis and selection for malignant phenotypes. Correspondingly, mutations that elevate mutation rates, producing a “mutator phenotype,” accelerate tumor formation. Heterozygous mutator alleles affecting the catalytic subunit of DNA polymerase (Pol) epsilon (encoded by the POLE gene) or bi-allelic mutations affecting mismatch repair (MMR) components, such as MSH2, arise frequently in a subset of colorectal and endometrial cancers. In some tumors, mutator alleles from both classes occur together and synergistically cause tremendous genetic instability. A key unanswered question is whether the severe mutation accumulation compromises tumor cell fitness and imposes a selection for “antimutator” phenotypes that allow the tumor to escape extinction. Evidence supporting the selection for antimutator alleles in strong mutator cells have been obtained using budding yeast, whose DNA replication and repair machinery are highly conserved with their mammalian counterparts. Our initial studies in haploid yeast show that mutations in the pol2 mutator alleles represent a major class of antimutator alleles. However, mutator suppression in diploid cells remains understudied. The strongest mutator allele known in human disease is POLE-P286R, which corresponds to pol2-P301R in yeast. We previously evolved diploid pol2-P301R/POL2 msh2Δ/msh2Δ mutator yeast strains to identify candidate antimutators that may arise in diploid cells. We also identified putative human antimutator alleles from The Cancer Genome Atlas database. Here, we test whether these alleles do indeed exert antimutator phenotypes by re-engineering them into diploid yeast. Our findings will provide a direct test of the relevance of antimutators for tumor evolution and define likely antimutator candidates for further study.

We have established a head and neck squamous cell carcinoma (HNSCC) cell line resource to facilitate translational research on HNSCC in individuals with Fanconi Anemia (FA). FA is a rare genetic disorder characterized by bone marrow failure and predisposition to leukemia and solid tumors. FA individuals have an extraordinarily high lifetime risk of HNSCC compared to the general population and the pathogenesis of these cancers is not understood. Apart from surgery, effective treatment of these cancers is limited by patient hypersensitivity to standard-of-care therapies that include ionizing radiation and DNA cross-linking drugs. Insight into the mechanistic origins of these cancers and the identification of less toxic, more effective therapies are necessary to improve survival and quality of life. The FA Cancer Cell Line Resource was developed to provide well-characterized, experimentally tractable pre-clinical models to investigate the origins, pathogenesis, treatment and prevention of FA HNSCC. Isogenic cell line pairs or trios included in this Resource were generated from FA patient-derived or sporadic HNSCC cells using CRISPR/Cas9 technology. The biochemical and molecular characteristics of these models were confirmed to verify the presence or absence of FA-associated phenotypes following gene edits. This Resource addresses the critical need for well-characterized and tractable disease models that are FA patient-derived, and versatile enough to permit both in vitro and in vivo analyses. These cell lines are now available at no cost to foster research through the Fanconi Anemia Research Fund-sponsored “Fanconi Anemia Research Materials” repository at Oregon Health and Sciences University (https://apps.ohsu.edu/research/fanconi-anemia/celllines.cfm).

Among the estimated 1.5 million adults and children living with HIV in Kenya, only 68% had suppressed viral loads in 2019. Improving adherence to antiretroviral therapy (ART) and minimizing ART failure is crucial in ensuring viral suppression. New types of viral load (VL) and drug resistance mutation (DRM) testing through point-of-care (POC) assays are potential solutions to optimize viral suppression rates. POC assays have faster turnaround times, can facilitate rapid clinical decision making, and are cost-effective, all of which can facilitate better patient outcomes. In the Opt4Kids and Opt4Mamas studies, children and pregnant/postpartum women either received the intervention, consisting of POC VL testing every three months with targeted DRM testing, or standard-of-care (SOC) testing according to national Kenyan guidelines. As undergraduate researchers, we are responsible for coding and analyzing 68 in-depth interviews collected from key informants and participants, including adolescents, children’s caregivers, and pregnant/postpartum women, to better understand how POC VL and targeted DRM testing influences viral suppression and may be utilized for use nation-wide. We used the socioecological model to identify the individual, interpersonal, organizational, societal/cultural, and structural/policy factors that influence viral suppression in children and pregnant/postpartum women. Through inductive coding and thematic analysis, we discovered six major domains of themes of interest amongst these populations: 1) HIV health literacy, 2) HIV testing and treatment experience, 3) differences between SOC VL testing and POC VL testing, and 4) DRM testing experience, 5) future improvements, and 6) impact of the COVID-19 pandemic on treatment and testing. Our findings will provide insight into ways of optimizing existing protocols and will determine whether our intervention is an improved alternative to current national standards. Ultimately, our research will shape national policies regarding HIV treatment and directly address ways to optimize HIV viral suppression.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the expression of virulence factors and public goods based on population density. P. aeruginosa QS consists in part of N-acyl homoserine lactone signal molecules that activate two separate regulatory proteins, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. The las and rhl regulons are hierarchical in lab strains, with LasR activating the transcription of rhlR; however, many pathogenic variants carry nonfunctional alleles of lasR and rely on rhlR as the dominant QS regulator. Two anti-activator proteins, QteE and QslA, restrict the expression of these two QS regulons; however, it is not clear how P. aeruginosa anti-activators function in many pathogenic strains. Identifying how anti-activators regulate QS in pathogenic variants could be crucial in developing therapies that do not rely on antibiotics. To investigate how QteE and QslA modulate QS, we overexpressed each anti-activator in P. aeruginosa and used transcriptional reporters to monitor the activity of rhlA, a RhlR regulated gene. Expression of both genes is reduced significantly in strains with over-expressed anti-activators. However, in a pathogenic variant, only over-expressing qteE delayed QS induction while over-expressing qslA had no effect. These results indicate that QteE can modulate QS by affecting LasR and RhlR levels, while QslA only modulates LasR levels. These experiments lay the foundation for therapeutic strategies centered on inhibiting QS rather than relying on conventional antibiotics.

Recent legalization of cannabis in various states has sparked research on the impact of cannabis use on postnatal outcomes. While previous research has yielded contradictory findings, some studies suggest increased cannabis use is directly associated with increased depression and psychological distress.We assert that prenatal mental health should be an essential consideration in the discourse surrounding prenatal cannabis use, as issues such as untreated maternal depression are risk factors for adverse postnatal outcomes like low birth weight and preterm delivery.However, few studies have considered the relationship between prenatal maternal mental health and cannabis use. Here we question: what is the association between cannabis use and psychological distress in pregnant individuals? For our sample population, we recruited pregnant individuals in the greater Seattle area. 12 individuals reported using cannabis (CB) at least 3 to 5 times a week throughout their first trimester of pregnancy, and 22 were non-cannabis users (n-CB). In early pregnancy, we administered the Brief Symptom Inventory (BSI), a self-reported measure, to evaluate psychological distress levels. We tracked cannabis use with weekly surveys from time of enrollment to birth, evaluating reasons for use and amount consumed, among other variables. We hypothesize that prenatal cannabis use will be associated with elevated BSI T-scores compared to the control group; within the prenatal cannabis use population, we expect cannabis use for mental health reasons to be associated with elevated BSI T-scores. More research is needed on possible causal versus correlational associations between cannabis use and psychological distress, and the role of psychosocial distress as a possible confounder in previous prenatal cannabis use and infant development studies.